Ethylene-responsive Element Binding Factor Research Articles

PhERF71 regulates petunia flower senescence by modulating ethylene biosynthesis

Ethylene, an endogenous hormone, commonly promotes flower senescence, but the molecular regulatory mechanisms underlying ethylene-induced flower senescence remain elusive. In this study, we describe an ethylene-responsive element binding factor, PhERF71, which affects flower senescence in petunia (Petunia hybrida). PhERF71 was up-regulated in senescing petunia flowers and after exogenous ethylene treatment, but silver thiosulfate (STS), an ethylene inhibitor, treatment decreased PhERF71 transcripts. Transient silencing of PhERF71 delayed petunia flower senescence, and reduced the transcription of senescence-associated gene 12 (PhSAG12). PhERF71-RNAi transgenic plants showed prolonged flower longevity, and PhERF71-overexpressing plants displayed shortened flower longevity, compared with wild-type (WT) plants. Electrolyte leakage rates and expression levels of PhSAG12 were lower in PhERF71-RNAi plants but higher in PhERF71-overexpressing plants. Silencing or overexpression of PhERF71 influenced the accumulation of ethylene, gibberellin (GA3), and abscisic acid (ABA), and the effect on ethylene accumulation was earlier than that on ABA and GA3. Ethylene treatment restored the delayed flower senescence to WT levels in PhERF71-silenced plants. Silencing of PhERF71 reduced expression levels of several ethylene biosynthetic genes, including 1-aminocyclopropane-1-carboxylic acid (ACC) synthase 4 (PhACS4), PhACS6, and ACC oxidase 4 (PhACO4), while overexpression of PhERF71 imposed the opposite effect on their expression levels. Dual luciferase assay showed that PhERF71 directly bound to the promoters of PhACS4 and PhACO4, and transient silencing of PhACS4 and PhACO4 delayed flower senescence. Our results suggest that PhERF71 is a positive regulator of flower senescence by promoting ethylene biosynthesis.

Genome-Wide Exploration of the Ethylene-Responsive Element-Binding Factor Gene Family in Sweet Cherry (Prunus avium L.): Preliminarily Unveiling Insights into Normal Development and Fruit Cracking

The ERF subfamily, a significant part of the APETALA2/ethylene-responsive element-binding factor (AP2/ERF) transcription family, plays a crucial role in plant growth, development, and stress responses. Despite its importance, research on this gene family in sweet cherry (Prunus avium L.) is limited. This study identified and analyzed the sweet cherry ERF subfamily in terms of classification, physicochemical properties, structural characteristics, chromosome distribution, gene replication and collinearity, Cis-acting elements, and potential protein interactions. Preliminary investigations of transcription during fruit cracking and normal development were also conducted. Fifty ERFs (PatiERF1~50) were identified, distributed unevenly across eight chromosomes and classified into ten groups with nineteen conserved motifs. Collinearity analysis with other plant species revealed homology, with the highest number of ERF orthologous genes found in apple (Malus domestica L.). Cis-acting elements, particularly abscisic acid response factor, were abundant in PatiERF promoters. Weighted gene co-expression network analysis (WGCNA) and quantitative real-time PCR (RT-qPCR) analysis indicated the involvement of PatiERFs in sweet cherry fruit development and cracking, and nine and four significant candidates related to these processes were speculated, respectively. Furthermore, four other classes of transcription factors (TFs), namely MYB, GRAS, BHLH, and BZIP, as well as 23 structure genes, were predicted to have co-expression and interaction relationships with PatiERFs during fruit development. This suggests their potential synergistic regulation with ERFs in the cherry fruit development process. Our study represents the first comprehensive genome-wide analysis of the ERF subfamily in sweet cherry, laying a crucial foundation for a deeper understanding of the molecular mechanisms correlated with fruit growth, development, and cracking mediated by ERF genes.

Enhancing genomic association studies in slash pine through close-range UAV-based morphological phenotyping.

In forestry genetics and industry, tree morphological traits such as height, crown size, and shape are critical for understanding growth dynamics and productivity. Traditional methods for measuring these traits are limited in efficiency, scalability, and accuracy, posing challenges for large-scale forest assessments. This study focuses on integrating unmanned aerial vehicle (UAV) technology with GWAS to improve genomic association studies in slash pine (Pinus elliottii). Seven key morphological traits have been identified (canopy area (CA), crown base height (CBH), crown length (CL), canopy volume (CV), crown width (CW), crown width height (CWH), and tree height (H)) through advanced UAV-based phenotyping. These associations account for a remarkable range of heritability in slash pine, with traits such as CBH, CL, CV, and H showing relatively high heritability across both Single nucleotide polymorphisms (SNP) and pedigree methods, indicating strong genetic influence, while traits such as CWH show lower heritability, suggesting greater environmental influence or non-additive genetic variance. The GWAS identified 28 associations, including 22 different SNPs localized to 16 candidate genes, that were significantly associated with the morphological traits of Slash Pine. Notably, two of these candidate genes, annotated as putative DEAD-like helicase and ethylene-responsive element binding factor (ERF), were present at different mutation sites and were significantly associated with CW and CA traits, respectively. These results demonstrate that the UAV imaging enables a comprehensive analysis of the Morphological growth response of slash pine and can facilitate the discovery of informative alleles to elucidate the genetic structure underlying complex phenotypic variation in conifers.

Alteration in the callogenesis, tropane alkaloid formation, and gene expression in Hyoscyamus niger under clinorotation.

This study aimed to investigate the effects of clinorotation induced by 2-D clinostat on the growth, tropane alkaloid production, gene expression, antioxidant capacity, and cellular defense responses in the callus tissue of Hyoscyamus niger. Callus induction was conducted by putting hypocotyl explants in the MS culture medium supplemented with 1 mgL-1 2,4-D and 1 mgL-1 BAP growth regulators. The sub-cultured calli were placed on a clinostat for 0, 3, 7, and 10 days (2.24 × 10-5 g on the edge of the callus ring). Clinorotation significantly increased callus fresh weight, dry weight, protein, carbohydrate, and proline contents compared to the control, and their maximum contents were obtained after 7 and 10 days. H2O2 level enhanced under clinorotation with a 76.3% rise after 10 days compared to control and positively affected the atropine (77.1%) and scopolamine (69.2%) productions. Hyoscyamine 6-beta hydroxylase and putrescine N-methyltransferase gene expression involved in the tropane alkaloid biosynthesis were upregulated markedly with 14.2 and 17.1-folds increase after 10 days of clinorotation, respectively. The expressions of jasmonic acid, mitogen-activated protein kinase, and ethylene-responsive element-binding transcription factor were upregulated, and the activity of peroxidase and catalase showed a 72.7 and 80% rise after 10 days. These findings suggest that microgravity can enhance callogenesis by stimulating the ROS level, which can impact the antioxidant enzymes, tropane alkaloid formation, and gene expression.

Transcript-wide identification and expression pattern analysis to comprehend the roles of AP2/ERF genes under development and abiotic stress in Trichosanthes kirilowii

BackgroundThe APETALA 2/ ethylene-responsive element binding factors (AP2/ERF), are thought to be associated with plant abiotic stress response, and involved in some plant hormone signaling pathways. Trichosanthes kirilowii is an important edible and medicinal crop, so far no research has been conducted on the TkAP2/ERF genes.ResultIn this study, a total of 135 TkERFs were identified, these genes were divided into 4 subfamilies and clustered into 13 groups. Moreover, 37 paralogous pairs were identified, with only two having Ka/Ks values greater than 1, proving that most TkERF genes underwent purifying selection during evolution. Co-expression networks constructed using transcriptome data at various flowering stages revealed that 50, 64, and 67 AP2/ERF genes correlated with members of the ethylene, gibberellin, and abscisic acid signaling pathways, respectively. When tissue cultured seedlings were treated with ETH, GA3 and ABA, 11, 12 and 17 genes were found to be up-regulated, respectively, suggesting that some members of the TkERF gene family may be involved in plant hormone signaling pathways. And under 4 ℃, PEG and NaCl treatment, 15, 20 and 19 genes were up-regulated, respectively, this suggested that these selected genes might be involved in plant abiotic stresses.ConclusionsOverall, we identified 135 AP2/ERF family members, a comprehensive analysis of AP2/ERF gene expression patterns by RNA-seq and qRT-PCR showed that they played important roles in flower development and abiotic stress. This study provided a theoretical basis for the functional study of TkAP2/ERF genes and the genetic improvement of T. kirilowii.

Single and co-inoculum of endophytic bacteria promote growth and yield of Jerusalem artichoke through upregulation of plant genes under drought stress.

Helianthus tuberosus L. (Jerusalem artichoke) produce inulin, a type of fructan, which possesses several biotechnology applications, e.g., sugar syrup, prebiotics, fiber in diabetic food, enabling blood sugar and cholesterol reduction. Drought reduces inulin accumulation in the tubers of Jerusalem artichoke as the plants protect themselves from this stress by induction of stress gene responses, effecting growth reduction. Endophytic bacteria are promising candidates to promote plant growth and yield particularly under abiotic stress. Therefore, three endophytic bacteria with plant growth promoting properties were examined for their ability to improve Jerusalem artichoke growth and yield under both well-watered and drought conditions when inoculated individually or in combinations in pot experiments with 2 factorial random complete block design. The interactions of the endophytic bacteria and plant host determined the differential gene expression in response to drought as revealed by quantitative polymerase chain reaction. Single inoculum of the endophytic bacteria increased the height, weight, root traits, and harvest index of Jerusalem artichoke compared to co-inocula under both well-watered and drought conditions. However, the co-inocula of Rossellomorea aquimaris strain 3.13 and Bacillus velezensis strain 5.18 proved to be a synergistic combination leading to high inulin accumulation; while the co-inocula of B. velezensis strain 5.18 and Micrococcus luteus strain 4.43 were not beneficial when used in combination. The genes, dehydrin like protein and ethylene responsive element binding factor, were upregulated in the plants inoculated with single inoculum and co-inocula of all endophytic bacteria during drought stress. Moreover, the gene expression of indole-3-acetic acid (IAA) amido synthetase were up-regulated in Jerusalem artichoke inoculated with M. luteus strain 4.43 during drought stress. The fructan:fructan 1-fructosyltransferase (1-FFT) was also stimulated by the endophytic bacteria particularly in drought condition; the results of this study could explain the relationship between endophytic bacteria and plant host for growth and yield promotion under well-watered and drought conditions.

RsERF40 contributes to cold stress tolerance and cell expansion of taproot in radish (Raphanus sativus L.).

The growth and development of taproots are inhibited by cold stress in radish (Raphanus sativus L.). Ethylene-responsive element binding factors (ERF) are key participators in the cold stress response and growth regulation of plants. However, the function of ERF genes in cold tolerance and root development in radish remains elusive. Here, we showed that the secondary growth of radish taproots was inhibited by cold stress. Comparative transcriptome analysis demonstrated that the RsERF40 gene is an important regulator of the cold stress response and root growth regulation. The cold tolerance of transgenic Arabidopsis plants overexpressing the RsERF40 gene was significantly improved. Overexpressing RsERF40 in the cold-sensitive radish genotype and silencing RsERF40 in the cold-tolerant radish genotype indicated that RsERF40 was beneficial for alleviating oxidative damage under cold stress in radish. Transgenic Arabidopsis seedlings showed an increase in the elongation and radial growth of dark-grown roots. RT-qPCR analysis showed that the expression of the cold-related genes (CORs) RsCOR78 and RsCOR413PM1 and the cell wall strengthening-related genes RsCESA6 and RsEXPB3 was upregulated in transgenic Arabidopsis seedlings. Yeast one-hybrid (Y1H) and dual-luciferase reporter assays (DLA) revealed that RsERF40 directly regulates RsCOR78, RsCOR413PM1, RsCESA6 and RsEXPB3 expression, illustrating that RsERF40 enhances cold tolerance and taproot growth by modulating osmotic adjustment and cell wall mechanical strength in radish. In this study, the RsERF40-regulon was firstly found to be a new cold response pathway independent of the CBF-COR pathway conferring cold stress tolerance with increasing radish taproot growth. These results provided novel insight into the molecular mechanism underlying cold stress response and would facilitate the genetic improvement of cold tolerance in radish and other root vegetable crops.

Os fatores de ligação do elemento responsivo ao etileno contribuem para a tolerância ao encharcamento, regulando os parâmetros fotossintéticos e fisiológicos na petúnia

ABSTRACT: Ethylene-responsive element binding factors (ERFs) are widely involved in the regulation of plant responses to different abiotic stresses. In petunia (Petunia × hybrida), PhERF2 belonging to the subfamily Ⅶ of ERF transcription factors participates in the response to waterlogging stress. In this study, we investigated waterlogging tolerance variation of WT and transgenic petunia plants with RNAi silencing and overexpression of PhERF2 through photosynthetic and physiological performance. Chlorophyll content and root vigor declined continuously in both WT and PhERF2 transgenic lines under waterlogging stress, but the extent of the fall in PhERF2-overexpressing lines was less than that in WT and PhERF2-RNAi lines. At the end of waterlogging treatment, soluble protein levels in PhERF2-overexpressing lines were significantly higher than those in WT and PhERF2-RNAi lines, while the latter showed a higher malondialdehyde content overall. Different degrees of reductions in Pn, Gs, and Tr levels occurred in both WT and PhERF2 transgenic lines upon exposure to waterlogging. The Ci levels of PhERF2-overexpressing lines decreased after 3 hours of waterlogging treatment, and the Ci levels of WT and PhERF2-RNAi lines gradually increased from 6 to 72 hours of waterlogging treatment. These data suggested that non-stomatal factors were the primary limiting factors for Pn in WT and PhERF2-RNAi lines under severe stress, while the stomatal opening was the main factor limiting Pn in PhERF2-overexpressing lines. Our results demonstrated that the contribution of PhERF2 to the waterlogging tolerance of petunia appears to depend on the regulation of physiological and photosynthetic responses. PhERF2 represents a hopeful candidate gene for enhancing waterlogging tolerance of ornamental plants.

VyUSPA3, a universal stress protein from the Chinese wild grape Vitis yeshanensis, confers drought tolerance to transgenic V. vinifera.

VyUSPA3 from the Chinese wild grape Vitis yeshanensis interacts with ERF105, PUB24 and NF-YB3, and overexpression of the VyUSPA3 gene in V. vinifera cv. 'Thompson Seedless' confers drought tolerance. Drought is a major abiotic stress factor that seriously affects the growth and yield of grapevine. Although many drought-related genes have been identified in Arabidopsis and other plants, the functions of only a few of their counterparts have been revealed in grape. Here, a universal stress protein (USP) A from the Chinese wild grape Vitis yeshanensis, VyUSPA3, was identified and its function was subsequently characterized by overexpressing or silencing the VyUSPA3 gene in V. vinifera cv. 'Thompson Seedless' via Agrobacterium-mediated genetic transformation. After 21 d of the drought treatment, most leaves of the untransformed (UT) 'Thompson Seedless' lines wilted, yet UT lines were less damaged compared to the RNAi-VyUSPA3 lines, nonetheless, the OE-VyUSPA3 lines were mostly unaffected. Meanwhile, OE-VyUSPA3 lines showed smaller stomatal aperture, more developed roots, higher leaf relative water content, proline content, and antioxidant enzyme activities, as well as lower malondialdehyde, H2O2 and O2•- accumulation than UT lines, but this response pattern was reversed in the RNAi-VyUSPA3 lines. Besides, the transcript levels of four drought-related genes (RD22, RD29B, DREB2A, and NCED1) in OE-VyUSPA3 lines were greater than those in the RNAi-VyUSPA3 and UT lines. In addition, a yeast two-hybrid assay and a bimolecular fluorescence complementation assay confirmed that VyUSPA3 interacted with ERF105, PUB24, and NF-YB3, respectively. This study revealed that VyUSPA3 improved drought tolerance in transgenic grapevines possibly through interaction with the hormone signaling, ubiquitination system, ethylene-responsive element binding factor and nuclear factors.

ERF4 interacts with and antagonizes TCP15 in regulating endoreduplication and cell growth in Arabidopsis.

Endoreduplication is prevalent during plant growth and development, and is often correlated with large cell and organ size. Despite its prevalence, the transcriptional regulatory mechanisms underlying the transition from mitotic cell division to endoreduplication remain elusive. Here, we characterize ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR 4 (ERF4) as a positive regulator of endoreduplication through its function as a transcriptional repressor. ERF4 was specifically expressed in mature tissues in which the cells were undergoing expansion, but was rarely expressed in young organs. Plants overexpressing ERF4 exhibited much larger cells and organs, while plants that lacked functional ERF4 displayed smaller organs than the wild-type. ERF4 was further shown to regulate cell size by controlling the endopolyploidy level in the nuclei. Moreover, ERF4 physically associates with the class I TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) protein TCP15, a transcription factor that inhibits endoreduplication by activating the expression of a key cell-cycle gene, CYCLIN A2;3 (CYCA2;3). A molecular and genetic analysis revealed that ERF4 promotes endoreduplication by directly suppressing the expression of CYCA2;3. Together, this study demonstrates that ERF4 and TCP15 function as a module to antagonistically regulate each other's activity in regulating downstream genes, thereby controlling the switch from the mitotic cell cycle to endoreduplication during leaf development. These findings expand our understanding of how the control of the cell cycle is fine-tuned by an ERF4-TCP15 transcriptional complex.

Simulated microgravity contributed to modification of callogenesis performance and secondary metabolite production in CannabisIndica

In vitro plant culture paves the way for meeting the industrial demand of pharmaceutically valuable secondary metabolites. This study intends to monitor how callus cells of Cannabis indica respond to the simulated microgravity (clinorotation; a Man-made technology). Callus initiation resulted from the culture of the leaf explant in a medium supplemented with kinetin (0.5 mgL−1) and 2, 4-D (2 mgL−1). Calli were treated with microgravity at three exposure times (0, 3, and 5 days). The microgravity treatments increased callus biomass about 2.5-fold. The clinorotation treatments transcriptionally induced the olivetolic acid cyclase (OAC) and olivetol synthase (OLS) genes about 6.2-fold. The tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes displayed a similar upward trend in response to microgravity. The applied treatments also stimulated the expression of the ethylene-responsive element-binding proteins (ERF1B) and WRKY1 transcription factors by an average of 7.6-fold. Moreover, the simulated microgravity triggered epigenetic modification in the DNA methylation profile. The HPLC-based assessment validated the high efficacy of the clinorotation treatments to increase the concentration of cannabinoids, including Cannabigerol (CBG) and Cannabidiol (CBD). However, the clinorotated calli contained a lower concentration of Tetrahydrocannabinol (THC) than the control group. The microgravity treatments increased concentrations of proline (79%), soluble sugars (61.3%), and proteins (21.4%) in calli. The biochemical assessment revealed that the clinorotation treatments slightly increased H2O2 concentration. The upregulation in the activities of peroxidase, catalase, and phenylalanine ammonia-lyase enzymes resulted from the microgravity treatments. Both HPLC and molecular assessments validated the significant efficacy of microgravity to enhance the production of cannabinoids.

VND-INTERACTING2 effectively inhibits transcriptional activities of VASCULAR-RELATED NAC-DOMAIN7 through a conserved sequence.

An Arabidopsis NAC domain transcription factor VND-INTERACTING2 (VNI2) was originally isolated as an interacting protein with another NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), a master regulator of xylem vessel element differentiation. VNI2 inhibits transcriptional activation activity of VND7 by forming a protein complex. Here, to obtain insights into how VNI2 regulates VND7, we tried to identify the amino acid region of VNI2 required for inhibition of VND7. VNI2 has an amino acid sequence similar to the ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR (ERF)-associated amphiphilic repression (EAR) motif, conserved in transcriptional repressors, at the C-terminus. A transient expression assay showed that the EAR-like motif of VNI2 was not required for inhibition of VND7. The C-terminal deletion series of VNI2 revealed that 10 amino acid residues, highly conserved in the VNI2 orthologs contributed to effective repression of the transcriptional activation activity of VND7. Observation of transgenic plants ectopically expressing VNI2 showed that the identified 10 amino acid sequence strongly affected xylem vessel formation and plant growth. These data indicated that the 10 amino acid sequence of VNI2 has an important role in its transcriptional repression activity and negative regulation of xylem vessel formation.

Genome-Wide Expression and Physiological Profiling of Pearl Millet Genotype Reveal the Biological Pathways and Various Gene Clusters Underlying Salt Resistance.

Pearl millet (Pennisetum glaucum L.) is a vital staple food and an important cereal crop used as food, feed, and forage. It can withstand heat and drought due to the presence of some unique genes; however, the mechanism of salt stress has been missing in pearl millet until now. Therefore, we conducted a comparative transcriptome profiling to reveal the differentially expressed transcripts (DETs) associated with salt stress in pearl millet at different time points, such as 1, 3, and 7 h, of salt treatment. The physiological results suggested that salt stress significantly increased proline, malondialdehyde (MDA) content, and hydrogen peroxide (H2O2) in pearl millet at 1, 3, and 7 h of salt treatment. In addition, pearl millet plants regulated the activities of superoxide dismutase, catalase, and peroxidase to lessen the impact of salinity. The transcriptomic results depicted that salt stress upregulated and downregulated the expression of various transcripts involved in different metabolic functions. At 1 and 7 h of salt treatment, most of the transcripts were highly upregulated as compared to the 3 h treatment. Moreover, among commonly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, the mitogen-activated protein kinase (MAPK) signaling pathway and peroxisome pathway were significantly enriched. The DETs related to hormone signaling (auxins, ethylene, gibberellin, and abscisic acid), kinases, protein modifications, and degradation were also identified, depicting the possible role of hormones and kinases to enhance plant tolerance against salt stress. Furthermore, the transcription factors, such as ethylene-responsive element binding factors (ERF), basic helix-loop-helix (bHLH), HMG box-containing protein (HBP), MADS, myeloblastosis (MYB), and WRKY, were predicted to significantly regulate different transcripts involved in salt stress responses at three different time points. Overall, this study will provide new insights to better understand the salt stress regulation mechanisms in pearl millet to improve its resistance against salinity and to identify new transcripts that control these mechanisms in other cereals.

Identification of Key Transcription Factors Related to Bacterial Spot Resistance in Pepper through Regulatory Network Analyses.

Bacterial spot (BS), caused by Xanthomonas campestris pv. Vesicatoria (Xcv), severely affects the quality and yield of pepper. Thus, breeding new pepper cultivars with enhanced resistance to BS can improve economic benefits for pepper production. Identification of BS resistance genes is an essential step to achieve this goal. However, very few BS resistance genes have been well characterized in pepper so far. In this study, we reanalyzed public multiple time points related to RNA-seq data sets from two pepper cultivars, the Xcv-susceptible cultivar ECW and the Xcv-resistant cultivar VI037601, post Xcv infection. We identified a total of 3568 differentially expressed genes (DEGs) between two cultivars post Xcv infection, which were mainly involved in some biological processes, such as Gene Ontology (GO) terms related to defense response to bacterium, immune system process, and regulation of defense response, etc. Through weighted gene co-expression network analysis (WGCNA), we identified 15 hub (Hub) transcription factor (TF) candidates in response to Xcv infection. We further selected 20 TFs from the gene regulatory network (GRN) potentially involved in Xcv resistance response. Finally, we predicted 4 TFs, C3H (p-coumarate 3-hydroxylase), ERF (ethylene-responsive element binding factor), TALE (three-amino-acid-loop-extension), and HSF (heat shock transcription factor), as key factors responsible for BS disease resistance in pepper. In conclusion, our study provides valuable resources for dissecting the underlying molecular mechanism responsible for Xcv resistance in pepper. Additionally, it also provides valuable references for mining transcriptomic data to identify key candidates for disease resistance in horticulture crops.

The Halophyte Halostachys caspica AP2/ERF Transcription Factor HcTOE3 Positively Regulates Freezing Tolerance in Arabidopsis.

The APETALA2 (AP2) and ethylene-responsive element-binding factor (ERF) gene family is one of the largest plant-specific transcription factor gene families, which plays a critical role in plant development and evolution, as well as response to various stresses. The TARGET OF EAT3 (TOE3) gene is derived from Halostachys caspica and belongs to the AP2 subfamily with two AP2 DNA-binding domains. Currently, AP2 family mainly plays crucial roles in plant growth and evolution, yet there are few reports about the role of AP2 in abiotic stress tolerance. Here, we report HcTOE3, a new cold-regulated transcription factor gene, which has an important contribution to freezing tolerance. The main results showed that the expression of HcTOE3 in the H. caspica assimilating branches was strongly induced by different abiotic stresses, including high salinity, drought, and extreme temperature (heat, chilling, and freezing), as well as abscisic acid and methyl viologen treatments. Overexpressing HcTOE3 gene (OE) induced transgenic Arabidopsis plant tolerance to freezing stress. Under freezing treatment, the OE lines showed lower content of malondialdehyde and electrolyte leakage and less accumulation of reactive oxygen species compared with the wild type. However, the survival rates, antioxidant enzyme activities, and contents of osmotic adjustment substance proline were enhanced in transgenic plants. Additionally, the OE lines increased freezing tolerance by up-regulating the transcription level of cold responsive genes (CBF1, CBF2, COR15, COR47, KIN1, and RD29A) and abscisic acid signal transduction pathway genes (ABI1, ABI2, ABI5, and RAB18). Our results suggested that HcTOE3 positively regulated freezing stress and has a great potential as a candidate gene to improve plant freezing tolerance.

MaMYB4, an R2R3-MYB Repressor Transcription Factor, Negatively Regulates the Biosynthesis of Anthocyanin in Banana.

Anthocyanins spatiotemporally accumulate in certain tissues of particular species in the banana plant, and MYB transcription factors (TFs) serve as their primary regulators. However, the precise regulatory mechanism in banana remains to be determined. Here, we report the identification and characterization of MaMYB4, an R2R3-MYB repressor TF, characterized by the presence of EAR (ethylene-responsive element binding factor–associated amphiphilic repression) and TLLLFR motifs. MaMYB4 expression was induced by the accumulation of anthocyanins. Transgenic banana plants overexpressing MaMYB4 displayed a significant reduction in anthocyanin compared to wild type. Consistent with the above results, metabolome results showed that there was a decrease in all three identified cyanidins and one delphinidin, the main anthocyanins that determine the color of banana leaves, whereas both transcriptome and reverse transcription–quantitative polymerase chain reaction analysis showed that many key anthocyanin synthesis structural genes and TF regulators were downregulated in MaMYB4 overexpressors. Furthermore, dual-luciferase assays showed that MaMYB4 was able to bind to the CHS, ANS, DFR, and bHLH promoters, leading to inhibition of their expression. Yeast two-hybrid analysis verified that MaMYB4 did not interact with bHLH, which ruled out the possibility that MaMYB4 could be incorporated into the MYB-bHLH-WD40 complex. Our results indicated that MaMYB4 acts as a repressor of anthocyanin biosynthesis in banana, likely due to a two-level repression mechanism that consists of reduced expression of anthocyanin synthesis structural genes and the parallel downregulation of bHLH to interfere with the proper assembly of the MYB-bHLH-WD40 activation complex. To the best of our knowledge, this is the first MYB TF that regulates anthocyanin synthesis that was identified by genetic methods in bananas, which will be helpful for manipulating anthocyanin coloration in banana programs in the future.

AtMPK6-induced phosphorylation of AtERF72 enhances its DNA binding activity and interaction with TGA4/OBF4 in Arabidopsis.

The ethylene-responsive element binding factor (ERF) family is a large family of transcription factors involved in plant development and environmental stress responses. We previously reported the identification of 29 putative substrates of Mitogen-activated Protein Kinase3 (AtMPK3), AtMPK4 and AtMPK6, based on a solid-phase phosphorylation screening using a lambda phage expression library in Arabidopsis thaliana. In this study, a putative MPK substrate, AtERF72 (At3g16770), was strongly phosphorylated by AtMPK6 on the serine residue at position 151 (Ser151). AtERF72 binds to the GCC box (AGCCGCC) in the promoters of several pathogenesis-related (PR) genes and activates their transcription. We also show that the DNA-binding activity of AtERF72 is enhanced upon phosphorylation by AtMPK6 in vitro. In addition, transient co-expression experiments in Arabidopsis protoplasts revealed that effector constructs expressing a mutant variant of AtERF72, AtERF72S151D (carrying a Ser to aspartic acid [Asp] substitution at amino acid position 151) showed higher expression of the β-glucuronidase (GUS) reporter gene driven by the GCC box element than effector constructs expressing the wild-type AtERF72. Furthermore, yeast two-hybrid assays revealed that the interaction between AtERF72S151D and TGA4/OBF4 was stronger than that between wild-type AtERF72 and TGA4/OBF4. Since AtERF72S151D is equivalent to AtERF72 phosphorylated by AtMPK6 at Ser151, these results suggest that the phosphorylation of AtERF72 by AtMPK6 triggers an event of transcriptional regulation from defence signalling in Arabidopsis.

A novel ethylene-responsive factor IbERF4 from sweetpotato negatively regulates abiotic stress

The ERF (ethylene-responsive element-binding factor) transcription factor family plays important roles in abiotic and biotic stress responses in plants. An EAR motif-containing ERF gene, IbERF4, was isolated from sweetpotato [Ipomoea batatas (L.) Lam]. IbERF4 was highly expressed in the early stage of storage roots and can be induced by various stresses. By overexpressing IbERF4 in Arabidopsis, the transgenic Arabidopsis showed greater sensitivity to drought and salt stress by down-regulating stress-related genes, such as rd22, rd29, AtCBF1 and AtCBF2. Overexpressing IbERF4 in sweetpotato made the transgenic sweetpotato more sensitive to salt stress and made propagating new seedlings more difficult. Consistently, the transcriptomic profiling results showed that genes involved in oxidative stress were significantly down-regulated in sweetpotato overexpression lines. These results indicated that IbERF4 plays as key regulators in sweetpotato abiotic stress.

Herbaspirillum rubrisubalbicans as a Phytopathogenic Model to Study the Immune System of Sorghum bicolor.

Herbaspirillum rubrisubalbicans is the causal agent of red stripe disease (RSD) and mottle stripe disease of sorghum and sugarcane, respectively. In all, 63 genotypes of Sorghum bicolor were inoculated with H. rubrisubalbicans, with 59 showing RSD symptoms. Quantitative trait loci (QTL) analysis in a recombinant inbred line (RIL) population identified several QTL associated with variation in resistance to RSD. RNA sequencing analysis identified a number of genes whose transcript levels were differentially regulated during H. rubrisubalbicans infection. Among those genes that responded to H. rubrisubalbicans inoculation were many involved in plant-pathogen interactions such as leucine-rich repeat receptors, mitogen-activated protein kinase 1, calcium-binding proteins, transcriptional factors (ethylene-responsive element binding factor), and callose synthase. Pretreatment of sorghum leaves with the pathogen-associated molecular pattern (PAMP) molecules flg22 and chitooctaose provided protection against subsequent challenge with the pathogen, suggesting that PAMP-triggered immunity plays an important role in the sorghum immunity response. These data present baseline information for the use of the genetically tractable H. rubrisubalbicans-sorghum pathosystem for the study of innate immunity and disease resistance in this important grain and bioenergy crop. Information gained from the use of this system is likely to be informative for other monocots, including those more intractable for experimental study (e.g., sugarcane).

Transcriptome profiling of high oleic peanut under low temperatureduring germination

High oleic (HO) peanut (Arachishypogaea L.) oils benefit human health and industrial production due to its superior nutritional composition and thermo-oxidative stability. However, HO peanut is sensitive to cold stress especially during germination, which limits its distribution in low temperature areas. To understand the molecular mechanism of cold responses in HO peanuts at germination stage, four HO peanut varieties with different cold tolerance were selected in field experiments to analyze their genome-wide gene regulation under low temperatures. High-throughput sequencing and transcriptome analysis revealed a total of 139 429 unigenes. Among these, 3520 common differentially expressed genes (DEG) were detected between two groups of cold-tolerant and cold-sensitive peanuts, and the number of up-regulated genes was greater than that of down-regulated genes in the cold-tolerant peanuts. Gene ontology analysis indicates that the number of DEGs involved in cell membrane metabolism and integrity as well as proteins located in the cell periphery were significantly higher in the cold-tolerant peanuts. KEGG pathway analysis suggests that plant-pathogen interaction and plant hormone signal transduction pathway play important roles in cold tolerance. Four cold-induced genes, TIC(TIME FOR COFFEE), ATX3(histone-lysine N-methyltransferase ATX3-like), AGO4(argonaute 4-like), FER(FERONIA-like receptor protein kinase), and three transcription factor genes, bHLH(bHLH49-like transcription factor), MYB(MYB-related protein 3R-1-like)and EREB(Ethylene-responsive element binding factor 6)were selected to verify the expression profile via real-time quantitative PCR detection. The expression of TIC, ATX3, AGO4, bHLH, MYB and EREB significantly increased within 3 hours after low temperature stress, while the expression of FER significantlyincreased after 12 hours, suggesting that these genes responded to low temperature stress during peanut germination. This work not only sheds light on the transcriptional regulation of HO peanut under low-temperature stress during germination but also provides data resources for screening candidate genes in improving peanuts stress resistance.