ERF-associated Amphiphilic Repression Research Articles

Rice transcriptional repressor OsTIE1 controls anther dehiscence and male sterility by regulating JA biosynthesis.

Proper anther dehiscence is essential for successful pollination and reproduction in angiosperms, and jasmonic acid (JA) is crucial for the process. However, the mechanisms underlying the tight regulation of JA biosynthesis during anther development remain largely unknown. Here, we demonstrate that the rice (Oryza sativa L.) ethylene-response factor-associated amphiphilic repression (EAR) motif-containing protein TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS (TCP) INTERACTOR CONTAINING EAR MOTIF PROTEIN1 (OsTIE1) tightly regulates JA biosynthesis by repressing TCP transcription factor OsTCP1/PCF5 during anther development. The loss of OsTIE1 function in Ostie1 mutants causes male sterility. The Ostie1 mutants display inviable pollen, early stamen filament elongation, and precocious anther dehiscence. In addition, JA biosynthesis is activated earlier and JA abundance is precociously increased in Ostie1 anthers. OsTIE1 is expressed during anther development, and OsTIE1 is localized in nuclei and has transcriptional repression activity. OsTIE1 directly interacts with OsTCP1, and overexpression of OsTCP1 caused early anther dehiscence resembling that of Ostie1. JA biosynthesis genes including rice LIPOXYGENASE are regulated by the OsTIE1-OsTCP1 complex. Our findings reveal that the OsTIE1-OsTCP1 module plays a critical role in anther development by finely tuning JA biosynthesis and provide a foundation for the generation of male sterile plants for hybrid seed production.

ETHYLENE RESPONSE FACTORS 4.1/4.2 with an EAR motif repress anthocyanin biosynthesis in red-skinned pears.

Red-skinned pears (Pyrus L.) are preferred to consumers for their attractive color and abundant anthocyanins. Pyrus ETHYLENE RESPONSE FACTOR 3 (PyERF3) positively regulates anthocyanin biosynthesis through interacting with Pyrus myeloblastosis family 114 (PyMYB114) and Pyrus basic helix-loop-helix 3 (PybHLH3) in red-skinned pears. However, the role of APETALA2/ethylene response factors (AP2/ERFs), which negatively regulate anthocyanin biosynthesis, remains unclear in red-skinned pears. Here, we validated that 2 AP2/ERFs, PyERF4.1 and PyERF4.2, screened from the transcriptome data of 'Starkrimson' pear (Pyrus communis L.) and its green mutant, inhibit anthocyanin biosynthesis in transgenic pear calli, as well as in overexpression and gene-edited tomato (Solanum lycopersicum) fruits. Meanwhile, the co-transformation of PyERF4.1/PyERF4.2 with PyERF3-PyMYB114-PybHLH3 inhibited anthocyanin biosynthesis in pear fruits and strawberry (Fragaria vesca) receptacles. Further assays showed that PyMYB114 activated the transcription of PyERF4.1/PyERF4.2; PyERF4.1/PyERF4.2 then interacted with PyERF3 to affect the stability of the PyERF3-PyMYB114-PybHLH3 complex, thereby inhibiting the transcription of the anthocyanin biosynthesis gene Pyrus anthocyanidin synthase (PyANS). Furthermore, deletion of the ERF-associated-amphiphilic repression (EAR) motif eliminated the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis, and a mutation of the PyERF4.2-EAR motif (LxLxM to LxLxL) strengthened the inhibitory effect, demonstrating that the EAR motif is indispensable for the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis in pears. Our study has shed light on a feedback regulatory loop mechanism that balances the excessive accumulation of anthocyanins in red-skinned pears, providing insights into the regulatory mechanism of anthocyanin biosynthesis and the regulatory network of coloration in red-skinned pears.

Arabidopsis TIE1 and TIE2 transcriptional repressors dampen cytokinin response during root development.

Cytokinin plays critical roles in root development. Cytokinin signaling depends on activation of key transcription factors known as type B Arabidopsis response regulators (ARRs). However, the mechanisms underlying the finely tuned regulation of type B ARR activity remain unclear. In this study, we demonstrate that the ERF-associated amphiphilic repression (EAR) motif–containing protein TCP interactor containing ear motif protein2 (TIE2) forms a negative feedback loop to finely tune the activity of type B ARRs during root development. Disruption of TIE2 and its close homolog TIE1 causes severely shortened roots. TIE2 interacts with type B ARR1 and represses transcription of ARR1 targets. The cytokinin response is correspondingly enhanced in tie1-1 tie2-1. We further show that ARR1 positively regulates TIE1 and TIE2 by directly binding to their promoters. Our findings demonstrate that TIEs play key roles in controlling plant development and reveal an important negative feedback regulation mechanism for cytokinin signaling.

Genome-wide investigation and expression analysis of the AP2/ERF family for selection of agarwood-related genes in Aquilaria sinensis (Lour.) Gilg.

Aquilaria sinensis is an important non-timber tree species for producing high-value agarwood, which is widely used as a traditional medicine and incense. Agarwood is the product of Aquilaria trees in response to injury and fungal infection. The APETALA2/ethylene responsive factor (AP2/ERF) transcription factors (TFs) play important roles in plant stress responses and metabolite biosynthesis. In this study, 119 AsAP2/ERF genes were identified from the A. sinensis genome and divided into ERF, AP2, RAV, and Soloist subfamilies. Their conserved motif, gene structure, chromosomal localization, and subcellular localization were characterized. A stress/defense-related ERF-associated amphiphilic repression (EAR) motif and an EDLL motif were identified. Moreover, 11 genes that were highly expressed in the agarwood layer in response to whole-tree agarwood induction technique (Agar-Wit) treatment were chosen, and their expression levels in response to methyl jasmonate (MeJA), salicylic acid (SA), or salt treatment were further analyzed using the quantitative real time PCR (qRT-PCR). Among the 11 genes, eight belonged to subgroup B-3. All 11 genes were significantly upregulated under salt treatment, while eight genes were significantly induced by both MeJA and SA. In addition, the gene clusters containing these upregulated genes on chromosomes were observed. The results obtained from this research not only provide useful information for understanding the functions of AP2/ERF genes in A. sinensis but also identify candidate genes and gene clusters to dissect their regulatory roles in agarwood formation for future research.

A C2H2-type zinc finger transcription factor, MdZAT17, acts as a positive regulator in response to salt stress

Salt stress restricts plant growth and productivity worldwide. Zinc finger proteins play important roles in response to various abiotic plant stresses. In this research, we identified and characterized the ZAT17 gene in Malus domestica, which encodes a C2H2-type zinc finger protein. MdZAT17 has two typical conserved zinc finger domains and an ERF-associated amphiphilic repression (EAR) motif. Promoter analysis showed that MdZAT17 contains several stress-related response elements (ABRE, CGTCA-motif, and TC-rich repeats), and qRT-PCR analysis showed that the expression level of MdZAT17 was induced by various abiotic stress treatments. The overexpression of MdZAT17 improved tolerance to salt stress in apple calli. The ectopic expression of MdZAT17 in Arabidopsis enhanced salt stress tolerance and led to lower malondialdehyde (MDA) content, lower reactive oxygen species (ROS) accumulation, and greater anthocyanin accumulation under salt stress. Moreover, the overexpression of MdZAT17 transgenic apple calli and Arabidopsis reduced the sensitivity to abscisic acid (ABA). In conclusion, our results indicate that MdZAT17 plays a positive regulatory role in salt tolerance, providing a theoretical basis for further research on its molecular mechanisms.

Cell death signalling is competitively but coordinately regulated by repressor-type and activator-type ethylene response factors in tobacco (Nicotiana tabacum) plants.

Ethylene response factors (ERFs) comprise one of the largest transcription factor families in many plant species. Tobacco (Nicotiana tabacum) ERF3 (NtERF3) and other ERF-associated amphiphilic repression (EAR) motif-containing ERFs are known to function as transcriptional repressors. NtERF3 and several repressor-type ERFs induce cell death in tobacco leaves and are also associated with a defence response against tobacco mosaic virus (TMV). We investigated whether transcriptional activator-type NtERFs function together with NtERF3 in the defence response against TMV infection by performing transient ectopic expression, together with gene expression, chromatin immunoprecipitation (ChIP) and promoter analyses. Transient overexpression of NtERF2 and NtERF4 induced cell death in tobacco leaves, albeit later than that induced by NtERF3. Fusion of the EAR motif to the C-terminal end of NtERF2 and NtERF4 abolished their cell death-inducing ability. The expression of NtERF2 and NtERF4 was upregulated at the early phase of N gene-triggered hypersensitive response (HR) against TMV infection. The cell death phenotype induced by overexpression of wild-type NtERF2 and NtERF4 was suppressed by co-expression of an EAR motif-deficient form of NtERF3. Furthermore, ChIP and promoter analyses suggested that NtERF2, NtERF3 and NtERF4 positively or negatively regulate the expression of NtERF3 by binding to its promoter region. Overall, our results revealed the cell death-inducing abilities of genes encoding activator-type NtERFs, including NtERF2 and NtERF4, suggesting that the HR-cell death signalling via the repressor-type NtERF3 is competitively but coordinately regulated by these NtERFs.

Full-length transcriptomic identification of R2R3-MYB family genes related to secondary cell wall development in Cunninghamia lanceolata (Chinese fir)

BackgroundR2R3-MYB is a class of transcription factor crucial in regulating secondary cell wall development during wood formation. The regulation of wood formation in gymnosperm has been understudied due to its large genome size. Using Single-Molecule Real-Time sequencing, we obtained full-length transcriptomic libraries from the developmental stem of Cunninghamia lanceolata, a perennial conifer known as Chinese fir. The R2R3-MYB of C. lanceolata (hereafter named as ClMYB) associated with secondary wall development were identified based on phylogenetic analysis, expression studies and functional study on transgenic line.ResultsThe evolutionary relationship of 52 ClMYBs with those from Arabidopsis thaliana, Eucalyptus grandis, Populus trichocarpa, Oryza sativa, two gymnosperm species, Pinus taeda, and Picea glauca were established by neighbour-joining phylogenetic analysis. A large number of ClMYBs resided in the woody-expanded subgroups that predominated with the members from woody dicots. In contrast, the woody-preferential subgroup strictly carrying the members of woody dicots contained only one candidate. The results suggest that the woody-expanded subgroup emerges before the gymnosperm/angiosperm split, while most of the woody-preferential subgroups are likely lineage-specific to woody dicots. Nine candidates shared the same subgroups with the A. thaliana orthologs, with known function in regulating secondary wall development. Gene expression analysis inferred that ClMYB1/2/3/4/5/26/27/49/51 might participate in secondary wall development, among which ClMYB1/2/5/26/27/49 were significantly upregulated in the highly lignified compression wood region, reinforcing their regulatory role associated with secondary wall development. ClMYB1 was experimentally proven a transcriptional activator that localised in the nucleus. The overexpression of ClMYB1 in Nicotiana benthamiana resulted in an increased lignin deposition in the stems. The members of subgroup S4, ClMYB3/4/5 shared the ERF-associated amphiphilic repression motif with AtMYB4, which is known to repress the metabolism of phenylpropanoid derived compounds. They also carried a core motif specific to gymnosperm lineage, suggesting divergence of the regulatory process compared to the angiosperms.ConclusionsThis work will enrich the collection of full-length gymnosperm-specific R2R3-MYBs related to stem development and contribute to understanding their evolutionary relationship with angiosperm species.

PtrMYB120 functions as a positive regulator of both anthocyanin and lignin biosynthetic pathway in a hybrid poplar.

Both anthocyanins and lignins are essential secondary metabolites in plant growth and development. Their biosynthesis is metabolically interconnected and diverges in the central metabolite 4-coumaroyl CoA of the phenylpropanoid pathway. Considerable progress has been made in understanding transcriptional regulation of genes involved in lignin and anthocyanin synthesis pathways, but the concerted regulation of these pathways is not yet fully understood. Here, we functionally characterized PtrMYB120, a R2R3-MYB transcription factor from Populus trichocarpa. Overexpression of PtrMYB120 in a hybrid poplar (i.e., 35S::PtrMYB120) was associated with increased anthocyanin (i.e., cyanidin 3-O-glucoside) accumulation and upregulation of anthocyanin biosynthetic genes. However, transgenic poplars with dominant suppression of PtrMYB120 function achieved by fusing the ERF-associated amphiphilic repression motif to PtrMYB120 (i.e., 35S::PtrMYB120-SRDX) had a dramatic decrease in not only anthocyanin but also Klason lignin content with downregulation of both anthocyanin and lignin biosynthetic genes. Indeed, 35S::PtrMYB120-SRDX poplars had irregularly shaped xylem vessels with reduced S-lignin content in stems, which was proportionally related to the level of the introduced PtrMYB120-SRDX gene. Furthermore, protoplast-based transcriptional activation assay using the PtrMYB120-GR system suggested that PtrMYB120 directly regulates genes involved in both anthocyanin and lignin biosynthesis, including chalcone synthase and ferulate-5 hydroxylase. Interestingly, the saccharification efficiency of line #6 of 35S::PtrMYB120-SRDX poplars, which had slightly reduced lignin content with a normal growth phenotype, was dramatically enhanced (>45%) by NaOH treatment. Taken together, our results suggest that PtrMYB120 functions as a positive regulator of both anthocyanin and lignin biosynthetic pathways and can be targeted to enhance saccharification efficiency in woody perennials.

BHLH IRIDOID SYNTHESIS 3 is a member of a bHLH gene cluster regulating terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Basic helix‐loop‐helix (bHLH) transcription factors (TFs) are key regulators of plant specialized metabolites, including terpenoid indole alkaloids (TIAs) in Catharanthus roseus. Two previously characterized subgroup‐IVa bHLH TFs, BIS1 (bHLH Iridoid Synthesis 1) and BIS2 regulate iridoid biosynthesis in the TIA pathway. We reanalyzed the recently updated C. roseus genome sequence and discovered that BIS1 and BIS2 are clustered on the same genomic scaffold with a previously uncharacterized bHLH gene, designated as BIS3. Only a few bHLH gene clusters have been studied to date. Comparative analysis of 49 genome sequences from different plant lineages revealed the presence of analogous bHLH clusters in core angiosperms, including the medicinal plants Calotropis gigantea (giant milkweed) and Gelsemium sempervirens (yellow jessamine), but not in the analyzed basal angiosperm and lower plants. Similar to the iridoid pathway genes, BIS3 is highly expressed in roots and induced by methyl jasmonate. BIS3 activates the promoters of iridoid branch genes, geraniol synthase (GES), geraniol 10‐hydroxylase (G10H), 8‐hydroxygeraniol oxidoreductase (8HGO), iridoid synthase (IS), 7‐deoxyloganetic acid glucosyl transferase (7‐DLGT), and 7‐deoxyloganic acid hydroxylase (7DLH), but not iridoid oxidase (IO). Transactivation of the promoters was abolished when BIS3 is converted to a dominant repressor by fusing with the ERF‐associated amphiphilic repression (EAR) sequence. In addition, BIS3 acts synergistically with BIS1 and BIS2 to activate the G10H promoter in tobacco cells. Mutation of the known bHLH TF binding motif, G‐box (CACGTG) in the G10H promoter significantly reduced but did not abolish the transactivation by BIS3. Promoter deletion analysis of G10H suggests that the sequences adjacent to the G‐box are also involved in the regulation by BIS3. Overexpression of BIS3 in C. roseus flower petals significantly upregulated the expression of iridoid biosynthetic genes and increased loganic acid accumulation. BIS2 expression was significantly induced by BIS3 although BIS3 did not directly activate the BIS2 promoter. Our results advance our understanding of the regulation of plant specialized metabolites by bHLH TF clusters.

Genome-wide analyses of direct target genes of an ERF11 transcription factor involved in plant defense against bacterial pathogens

Ethylene responsive factor ERF11 containing the ERF-associated amphiphilic repression (EAR) motif enhances plant resistance to bacterial pathogens. However, the underlying molecular mechanisms regulated by transcription factor ERF11 are poorly understood, in tobacco or other model plants. Here, we revealed the genome-wide binding landscape of BrERF11b in Nicotiana benthamian by conducting chromatin immunoprecipitation experiments followed by high-throughput sequencing (ChIP-seq) and bioinformatic analyses. Our results also revealed a GCCbox-like consensus BrERF11b-binding DNA motif: VCGCCGCC. By further integrative analysis of ChIP-seq and RNA-seq data, and the confirmation of electrophoretic mobility shift assay (EMSA), we screened three direct target genes NbNIMIN2, NbTAF15b and NbERF4. These results suggest that ERF11 may be involved in NPR1-mediated systemic acquired resistance (SAR), nucleotide-binding leucine-rich repeat immune receptors (NLR) -mediated autoimmunity, and H2O2 generation, by direct transcriptional repression of NIM1-INTERACTING2 (NIMIN2), and transcriptional activation of TATA-binding protein-associated factor 15b (TAF15b) and ERF4. Our findings provide insightful information and valuable gene resource in unraveling the regulatory networks of plant defense responses to bacterial pathogens.

Arabidopsis ZINC FINGER PROTEIN1 Acts Downstream of GL2 to Repress Root Hair Initiation and Elongation by Directly Suppressing bHLH Genes.

Cys2His2-like fold group (C2H2)-type zinc finger proteins promote root hair growth and development by regulating their target genes. However, little is known about their potential negative roles in root hair initiation and elongation. Here, we show that the C2H2-type zinc finger protein named ZINC FINGER PROTEIN1 (AtZP1), which contains an ERF-associated amphiphilic repression (EAR) motif, negatively regulates Arabidopsis (Arabidopsis thaliana) root hair initiation and elongation. Our results demonstrate that AtZP1 is highly expressed in root hairs and that AtZP1 inhibits transcriptional activity during root hair development. Plants overexpressing AtZP1 lacked root hairs, while loss-of-function mutants had longer and more numerous root hairs than the wild type. Transcriptome analysis indicated that AtZP1 downregulates genes encoding basic helix-loop-helix (bHLH) transcription factors associated with root hair cell differentiation and elongation. Mutation or deletion of the EAR motif substantially reduced the inhibitory activity of AtZP1. Chromatin immunoprecipitation assays, AtZP1:glucocorticoid receptor (GR) induction experiments, electrophoretic mobility shift assays, and yeast one-hybrid assays showed that AtZP1 directly targets the promoters of bHLH transcription factor genes, including the key root hair initiation gene ROOT HAIR DEFECTIVE6 (RHD6) and root hair elongation genes ROOT HAIR DEFECTIVE 6-LIKE 2 (RSL2) and RSL4, and suppresses root hair development. Our findings suggest that AtZP1 functions downstream of GL2 and negatively regulates root hair initiation and elongation, by suppressing RHD6, RSL4, and RSL2 transcription via the GL2/ZP1/RSL pathway.

Transcriptional network regulation of the brassinosteroid signaling pathway by the BES1-TPL-HDA19 co-repressor complex.

The brassinosteroid-related BES1 and BZR1 transcription factors dynamically modulate downstream gene networks via the TPL-HDA19 co-repressor complex in BR-signaling pathways in Arabidopsis thaliana. Brassinosteroids (BRs) are plant steroid hormones that are essential for diverse growth and developmental processes across the whole life cycle of plants. In Arabidopsis thaliana, the BR-related transcription factors BRI1-EMS-SUPPRESSOR 1 (BES1) and BRASSINAZOLE-RESISTANT 1 (BZR1) regulate a range of global gene expression in response to BR and several external signaling cues; however, the molecular mechanisms by which they mediate the reprogramming of downstream transcription remain unclear. We here report that formation of a protein complex between BES1 and BZR1 and Histone Deacetylase 19 (HDA19) via the conserved ERF-associated amphiphilic repression (EAR) motif proved essential for regulation of BR-signaling-related gene expression. Defects in BR-related functions of BES1 and BZR1 proteins containing a mutated EAR motif were completely rescued by artificial fusion with EAR-repression domain (SRDX), TOPLESS (TPL), or HDA19 proteins. RNA-sequencing analysis of Arabidopsis plants over-expressing bes1-DmEAR or bes1-DmEAR-HDA19 revealed an essential role for HDA19 activity in regulation of BES1/BZR1-mediated BR signaling. In addition to BR-related gene expression, the BES1-HDA19 transcription factor complex was important for abiotic stress-related drought stress tolerance and organ boundary formation. These results suggested that integrating activation of BR-signaling pathways with the formation of the protein complex containing BES1/BZR1 and TPL-HDA19 via the EAR motif was important in fine-tuning BR-related gene networks in plants.

The R2R3-MYB Transcription Factor MYB49 Regulates Cadmium Accumulation.

Abscisic acid (ABA) reduces accumulation of potentially toxic cadmium (Cd) in plants. How the ABA signal is transmitted to modulate Cd uptake remains largely unclear. Here, we report that the basic region/Leu zipper transcription factor ABSCISIC ACID-INSENSITIVE5 (ABI5), a central ABA signaling molecule, is involved in ABA-repressed Cd accumulation in plants by physically interacting with a previously uncharacterized R2R3-type MYB transcription factor, MYB49. Overexpression of the Cd-induced MYB49 gene in Arabidopsis (Arabidopsis thaliana) resulted in a significant increase in Cd accumulation, whereas myb49 knockout plants and plants expressing chimeric repressors of MYB49:ERF-associated amphiphilic repression motif repression domain (SRDX49) exhibited reduced accumulation of Cd. Further investigations revealed that MYB49 positively regulates the expression of the basic helix-loop-helix transcription factors bHLH38 and bHLH101 by directly binding to their promoters, leading to activation of IRON-REGULATED TRANSPORTER1, which encodes a metal transporter involved in Cd uptake. MYB49 also binds to the promoter regions of the heavy metal-associated isoprenylated plant proteins (HIPP22) and HIPP44, resulting in up-regulation of their expression and subsequent Cd accumulation. On the other hand, as a feedback mechanism to control Cd uptake and accumulation in plant cells, Cd-induced ABA up-regulates the expression of ABI5, whose protein product interacts with MYB49 and prevents its binding to the promoters of downstream genes, thereby reducing Cd accumulation. Our results provide new insights into the molecular feedback mechanisms underlying ABA signaling-controlled Cd uptake and accumulation in plants.

Impact of Alternatively Polyadenylated Isoforms of ETHYLENE RESPONSE FACTOR4 with Activator and Repressor Function on Senescence in Arabidopsis thaliana L.

Leaf senescence is highly regulated by transcriptional reprogramming, implying an important role for transcriptional regulators. ETHYLENE RESPONSE FACTOR4 (ERF4) was shown to be involved in senescence regulation and to exist in two different isoforms due to alternative polyadenylation of its pre-mRNA. One of these isoforms, ERF4-R, contains an ERF-associated amphiphilic repression (EAR) motif and acts as repressor, whereas the other form, ERF4-A, is lacking this motif and acts as activator. Here, we analyzed the impact of these isoforms on senescence. Both isoforms were able to complement the delayed senescence phenotype of the erf4 mutant with a tendency of ERF4-A for a slightly better complementation. However, overexpression led to accelerated senescence of 35S:ERF4-R plants but not of 35S:ERF4-A plants. We identified CATALASE3 (CAT3) as direct target gene of ERF4 in a yeast-one-hybrid screen. Both isoforms directly bind to the CAT3 promoter but have antagonistic effects on gene expression. The ratio of ERF4-A to ERF4-R mRNA changed during development, leading to a complex age-dependent regulation of CAT3 activity. The RNA-binding protein FPA shifted the R/A-ratio and fpa mutants are pointing towards a role of alternative polyadenylation regulators in senescence.

Genome-wide identification of AP2/ERF superfamily genes and their expression during fruit ripening of Chinese jujube

The Ethylene response factor (ERF) belongs to the APETALA2/ethylene response factor (AP2/ERF) superfamily, located at the end of the ethylene signalling pathway, and has important roles in regulating the ethylene-related response genes. Thus, identifying and charactering this transcription factor would be helpful to elucidate ethylene related fruit ripening regulation in Chinese jujube (Ziziphus jujuba Mill.). In the present study, 119 AP2/ERF genes, including 5 Related to ABI3/VPs (RAV), 17 AP2s, 57 ERFs, 39 dehydration-responsive element-binding (DREB) factors and 1 soloist gene, were identified from the jujube genome sequences. Genome localization, gene duplication, phylogenetic relationships and conserved motifs were simultaneously analysed. Using available transcriptomic data, 85 genes with differential transcripts in the flower, leaf and fruit were detected, suggesting a broad regulation of AP2/ERF genes in the growth and development of jujube. Among them, 44 genes were expressed in the fruit. As assessed by quantitative PCR, 15 up- and 23 downregulated genes corresponding to fruit full maturity were found, while in response to 100 μl l−1 ethylene, 6 up- and 16 downregulated genes were generated. By comparing the output, ZjERF54 and DREB39 were found to be the best candidate genes that positively participated in jujube fruit ripening, while ZjERF25 and ZjERF36, which had an ERF-associated amphiphilic repression (EAR) motif, were ripening repressors. These findings help to gain insights into AP2/ERF gene evolution and provide a useful resource to further understand the ethylene regulatory mechanisms underlying Chinese jujube fruit ripening.

Arabidopsis ETHYLENE RESPONSE FACTOR 8 (ERF8) has dual functions in ABA signaling and immunity

BackgroundETHYLENE RESPONSE FACTOR (ERF) 8 is a member of one of the largest transcription factor families in plants, the APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) superfamily. Members of this superfamily have been implicated in a wide variety of processes such as development and environmental stress responses.ResultsIn this study we demonstrated that ERF8 is involved in both ABA and immune signaling. ERF8 overexpression induced programmed cell death (PCD) in Arabidopsis and Nicotiana benthamiana. This PCD was salicylic acid (SA)-independent, suggesting that ERF8 acts downstream or independent of SA. ERF8-induced PCD was abolished by mutations within the ERF-associated amphiphilic repression (EAR) motif, indicating ERF8 induces cell death through its transcriptional repression activity. Two immunity-related mitogen-activated protein kinases, MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) and MPK11, were identified as ERF8-interacting proteins and directly phosphorylated ERF8 in vitro. Four putative MPK phosphorylation sites were identified in ERF8, one of which (Ser103) was determined to be the predominantly phosphorylated residue in vitro, while mutation of all four putative phosphorylation sites partially suppressed ERF8-induced cell death in N. benthamiana. Genome-wide transcriptomic analysis and pathogen growth assays confirmed a positive role of ERF8 in mediating immunity, as ERF8 knockdown or overexpression lines conferred compromised or enhanced resistance against the hemibiotrophic bacterial pathogen Pseudomonas syringae, respectively.ConclusionsTogether these data reveal that the ABA-inducible transcriptional repressor ERF8 has dual roles in ABA signaling and pathogen defense, and further highlight the complex influence of ABA on plant-microbe interactions.

Genome-wide identification and characterization of WUSCHEL-related homeobox (WOX) genes in Salix suchowensis

Members of the WUSCHEL-related homeobox (WOX) transcription factor family are essential for determining cell fate and regulating diverse developmental processes in plants. Many WOX genes have been systematically investigated in woody plants such as Populus trichocarpa, but not in Salix suchowensis. Whole-genome sequence data for S. suchowensis is now available for comprehensive study of WOX genes in S. suchowensis. We thus surveyed the genome of S. suchowensis and demonstrated active expression of 15 WOX genes. In a phylogenetic analysis of WOX genes, the 15 SsWOX genes clustered among the modern/WUS, intermediate and ancient clades similar to the WOX genes of Arabidopsis thaliana. Based on the conserved intron/exon structure, SsWOX genes in the same subgroup had similar conserved exon–intron structures and motif domains. Furthermore, among several SsWOX subgroups, WUS (Wuschel)-box and EAR (the ERF-associated amphiphilic repression)-like motifs were conserved. Expression profiles of WOX genes in roots, stems and leaves indicate that SsWOX genes have various conserved roles in the tissues. Comparative analysis of the expression patterns in Salix suchowensis with that of Arabidopsis suggests that different shoot regeneration abilities are controlled by different WOX genes in plants. The analysis provide an overview of differentially expressed SsWOX genes during shoot regeneration, but also contribute to understanding the evolution of WOX genes in Salicaceae and the interrelations of WOX genes and other transcription factors, providing targets for further study.

TOPLESS mediates brassinosteroid control of shoot boundaries and root meristem development in Arabidopsis thaliana.

The transcription factor BRI1-EMS-SUPRESSOR 1 (BES1) is a master regulator of brassinosteroid (BR)-regulated gene expression. BES1 together with BRASSINAZOLE-RESISTANT 1 (BZR1) drive activated or repressed expression of several genes, and have a prominent role in negative regulation of BR synthesis. Here, we report that BES1 interaction with TOPLESS (TPL), via its ERF-associated amphiphilic repression (EAR) motif, is essential for BES1-mediated control of organ boundary formation in the shoot apical meristem and the regulation of quiescent center (QC) cell division in roots. We show that TPL binds via BES1 to the promoters of the CUC3 and BRAVO targets and suppresses their expression. Ectopic expression of TPL leads to similar organ boundary defects and alterations in QC cell division rate to the bes1-d mutation, while bes1-d defects are suppressed by the dominant interfering protein encoded by tpl-1, with these effects respectively correlating with changes in CUC3 and BRAVO expression. Together, our data unveil a pivotal role of the co-repressor TPL in the shoot and root meristems, which relies on its interaction with BES1 and regulation of BES1 target gene expression.

Differential isoform expression and protein localization from alternatively spliced Apetala2 in peanut under drought stress

APETALA2 (AP2) belongs to the AP2/Ethylene Responsive Factor (ERF) family and regulates expression levels of downstream stress responsive genes as a transcription factor. In this study, we cloned six different isoforms of AhAP2 from peanut (Arachis hypogaea). Four isoforms (AhAP2.1, AhAP2.2, AhAP2.3 and AhAP2.4) had both AP2/ERF DNA binding domains and ERF-associated amphiphilic repression (EAR) motifs. Two isoforms (AhAP2.5 and AhAP2.6) only had an EAR suppressor domain. After agroinfiltration, AhAP2.1, AhAP2.3, and AhAP2.4 fused to yellow fluorescent protein (YFP) showed localization to the nucleolus, which is the site of transcription and ribosome biogenesis. AhAP2.2-YFP showed a dispersed signal in the nucleus. AhAP2.5 and AhAP2.6 fused to YFP localized to both the nucleus and cytoplasm. In addition, increased levels of AhAP2.1 and AhAP2.2 transcripts were observed in drought-treated peanut leaves, suggesting differential transcriptional regulation under drought stress conditions.

Papaya CpERF9 acts as a transcriptional repressor of cell-wall-modifying genes CpPME1/2 and CpPG5 involved in fruit ripening.

CpERF9 controls papaya fruit ripening through transcriptional repression of cell-wall-modifying genes CpPME1/2 and CpPG5 by directly binding to their promoters. Papaya fruit ripening is an intricate and highly coordinated developmental process which is controlled by the action of ethylene and expression of numerous ethylene-responsive genes. Ethylene response factors (ERFs) representing the last regulators of ethylene-signaling pathway determine the specificities of ethylene response. However, knowledge concerning the transcriptional controlling mechanism of ERF-mediated papaya fruit ripening is limited. In the present work, a gene-encoding AP2/ERF protein with two ERF-associated amphiphilic repression (EAR) motifs, named CpERF9, was characterized from papaya fruit. CpERF9 was found to localize in nucleus, and possess transcriptional repression ability. CpERF9 expression steadily decreased during papaya fruit ripening, while several genes encoding pectin methylesterases (PMEs) and polygalacturonases (PGs), such as CpPME1/2 and CpPG5, were gradually increased, paralleling the decline of fruit firmness. Electrophoretic mobility shift assay (EMSA) demonstrated a specific binding of CpERF9 to promoters of CpPME1/2 and CpPG5, via the GCC-box motif. Transient expression of CpERF9 in tobacco repressed CpPME1/2 and CpPG5 promoter activities, which was depended on two EAR motifs of CpERF9 protein. Taken together, these findings suggest that papaya CpERF9 may act as a transcriptional repressor of several cell-wall modifying genes, such as CpPME1/2 and CpPG5, via directly binding to their promoters.