GCC Box Research Articles

Arg156 in the AP2-Domain Exhibits the Highest Binding Activity among the 20 Individuals to the GCC Box in BnaERF-B3-hy15, a Mutant ERF Transcription Factor from Brassica napus.

To develop mutants of the ERF factor with more binding activities to the GCC box, we performed in vitro directed evolution by using DNA shuffling and screened mutants through yeast one-hybrid assay. Here, a series of mutants were obtained and used to reveal key amino acids that induce changes in the DNA binding activity of the BnaERF-B3 protein. With the BnaERF-B3-hy15 as the template, we produced 12 mutants which host individual mutation of potential key residues. We found that amino acid 156 is the key site, and the other 18 mutants host the 18 corresponding individual amino acid residues at site 156. Among the 20 individuals comprising WT (Gly156), Mu3 (Arg156), and 18 mutants with other 18 amino acid residues, Arg156 in the AP2-domain is the amino acid residue with the highest binding activity to the GCC box. The structure of the α-helix in the AP2-domain affects the binding activity. Other residues within AP2-domain modulated binding activity of ERF protein, suggesting that these positions are important for binding activity. Comparison of the mutant and wild-type transcription factors revealed the relationship of protein function and sequence modification. Our result provides a potential useful resource for understanding the trans-activation of ERF proteins.

An Ethylene-responsive Factor BpERF11 Negatively Modulates Salt and Osmotic Tolerance in Betula platyphylla.

Ethylene responsive factors (ERFs) play important roles in the abiotic stress; however, only a few ERF genes from woody plants have been functionally characterized. In the present study, an ERF gene from Betula platyphylla (birch), BpERF11, was functionally characterized in response to abiotic stress. BpERF11 is a nuclear protein, which could specifically bind to GCC boxes and DRE motifs. BpERF11-overexpressing and BpERF11 RNA interference (RNAi) knockdown plants were generated for gain- and loss-of-function analysis. BpERF11 negatively regulates resistance to salt and severe osmotic stress, and the transgenic birch plants overexpressing BpERF11 shows increased electrolyte leakage and malondialdehyde (MDA) contents. BpERF11 inhibits the expression of an AtMYB61 homologous gene, resulting in increased stomatal aperture, which elevated the transpiration rate. Furthermore, BpERF11 downregulates the expression of P5CS, SOD and POD genes, but upregulates the expression of PRODH and P5CDH, which results in reduced proline levels and increased reactive oxygen species (ROS) accumulation. BpERF11 also significantly inhibits the expression of LEA and dehydrin genes that involve in abiotic stress tolerance. Therefore, BpERF11 serves as a transcription factor that negatively regulates salt and severe osmotic tolerance by modulating various physiological processes.

Structural modeling and molecular simulation analysis of HvAP2/EREBP from barley

AP2/ERF transcription factors play a critical role in plant development and stress adaptation. This study reports the three-dimensional ab initio-based model of AP2/EREBP protein of barley and its interaction with DNA. Full-length coding sequence of HvAP2/EREBP gene isolated from two Indian barley cultivars, RD 2503 and RD 31, was used to model the protein. Of five protein models obtained, the one with lowest C-score was chosen for further analysis. The N- and C-terminal regions of HvAP2 protein were found to be highly disordered. The dynamic properties of AP2/EREBP and its interaction with DNA were investigated by molecular dynamics simulation. Analysis of trajectories from simulation yielded the equilibrated conformation between 2-10ns for protein and 7-15ns for protein-DNA complex. We established relationship between DNA having GCC box and DNA-binding domain of HvAP2/EREBP was established by modeling 11-base-pair-long nucleotide sequence and HvAP2/EREBP protein using ab initio method. Analysis of protein–DNA interaction showed that a β-sheet motif constituting amino acid residues THR105, ARG100, ARG93, and ARG83 seems to play important role in stabilizing the complex as they form strong hydrogen bond interactions with the DNA motif. Taken together, this study provides first-hand comprehensive information detailing structural conformation and interactions of HvAP2/EREBP proteins in barley. The study intensifies the role of computational approaches for preliminary examination of unknown proteins in the absence of experimental information. It also provides molecular insight into protein–DNA binding for understanding and enhancing abiotic stress resistance for improving the water use efficiency in crop plants.

PsAP2 an AP2/ERF family transcription factor from Papaver somniferum enhances abiotic and biotic stress tolerance in transgenic tobacco.

The AP2/ERFs are one of the most important family of transcription factors which regulate multiple responses like stress, metabolism and development in plants. We isolated PsAP2 a novel AP2/ERF from Papaver somniferum which was highly upregulated in response to wounding followed by ethylene, methyl jasmonate and ABA treatment. PsAP2 showed specific binding with both DRE and GCC box elements and it was able to transactivate the reporter genes in yeast. PsAP2 overexpressing transgenic tobacco plants exhibited enhanced tolerance towards both abiotic and biotic stresses . Real time transcript expression analysis showed constitutive upregulation of tobacco Alternative oxidase1a and Myo-inositol-1-phosphate synthase in PsAP2 overexpressing tobacco plants. Further, PsAP2 showed interaction with NtAOX1a promoter in vitro, it also specifically activated the NtAOX1a promoter in yeast and tobacco BY2 cells. The silencing of PsAP2 using VIGS lead to significant reduction in the AOX1 level in P. somniferum. Taken together PsAP2 can directly bind and transcriptionally activate NtAOX1a and its overexpression in tobacco imparted increased tolerance towards both abiotic and biotic stress.

Ethylene Response Factors: A Key Regulatory Hub in Hormone and Stress Signaling.

Ethylene is essential for many developmental processes and a key mediator of biotic and abiotic stress responses in plants. The ethylene signaling and response pathway includes Ethylene Response Factors (ERFs), which belong to the transcription factor family APETALA2/ERF. It is well known that ERFs regulate molecular response to pathogen attack by binding to sequences containing AGCCGCC motifs (the GCC box), a cis-acting element. However, recent studies suggest that several ERFs also bind to dehydration-responsive elements and act as a key regulatory hub in plant responses to abiotic stresses. Here, we review some of the recent advances in our understanding of the ethylene signaling and response pathway, with emphasis on ERFs and their role in hormone cross talk and redox signaling under abiotic stresses. We conclude that ERFs act as a key regulatory hub, integrating ethylene, abscisic acid, jasmonate, and redox signaling in the plant response to a number of abiotic stresses.

Isolation and molecular characterization of a novel WIN1/SHN1 ethylene-responsive transcription factor TdSHN1 from durum wheat (Triticum turgidum. L. subsp. durum).

Over the last decade, APETALA2/Ethylene Responsive Factor (AP2/ERF) proteins have become the subject of intensive research activity due to their involvement in a variety of biological processes. This research led to the identification of AP2/ERF genes in many species; however, little is known about these genes in durum wheat, one of the most important cereal crops in the world. In this study, a new member of the AP2/ERF transcription factor family, designated TdSHN1, was isolated from durum wheat using thermal asymetric interlaced PCR (TAIL-PCR) method. Protein sequence analysis showed that TdSHN1 contained an AP2/ERF domain of 63 amino acids and a putative nuclear localization signal (NLS). Phylogenetic analysis showed that TdSHN1 belongs to a group Va protein in the ERF subfamily which contains the Arabidopsis ERF proteins (SHN1, SHN2, and SHN3). Expression of TdSHN1 was strongly induced by salt, drought, abscisic acid (ABA), and cold. In planta, TdSHN1 protein was able to activate the transcription of GUS reporter gene driven by the GCC box and DRE element sequences. In addition, TdSHN1 was targeted to the nucleus when transiently expressed in tobacco epidermal cells. In transgenic yeast, overexpression of TdSHN1 increased tolerance to multiple abiotic stresses. Taken together, the results showed that TdSHN1 encodes an abiotic stress-inducible, transcription factor which confers abiotic stress tolerance in yeast. TdSHN1 is therefore a promising candidate for improvement of biotic and abiotic stress tolerance in wheat as well as other crops.

The Potato ERF Transcription Factor StERF3 Negatively Regulates Resistance to Phytophthora infestans and Salt Tolerance in Potato.

Ethylene response factors (ERFs) are unique to the plant kingdom and play crucial roles in plant response to various biotic and abiotic stresses. We show here that a potato StERF3, which contains an ERF-associated amphiphilic repression (EAR) motif in its C-terminal region, negatively regulates resistance to Phytophthora infestans and salt tolerance in potato. The StERF3 promoter responds to induction by salicylic acid, ABA ethylene and NaCl, as well as P. infestans, the causal agent of potato late blight disease. StERF3 could bind to the GCC box element of the HIS3 promoter and activate transcription of HIS3 in yeast cells. Importantly, silencing of StERF3 in potato produced an enhanced foliage resistance to P. infestans and elevated plant tolerance to NaCl stress accompanied by the activation of defense-related genes (PR1, NPR1 and WRKY1). In contrast, StERF3-overexpressing plants showed reduced expression of these defense-related genes and enhanced susceptibility to P. infestans, suggesting that StERF3 functions as a negative regulator of downstream defense- and/or stress-related genes in potato. StERF3 is localized to the nucleus. Interestingly, yeast two-hybrid assay and a bimolecular fluorescence complementation (BiFC) test clarified that StERF3 could interact with other proteins in the cytoplasm which may lead to its re-localization between the nucleus and cytoplasm, revealing a novel means of StERF3 regulation. Taken together, these data provide new insights into the mechanism underlying how StERF3 negatively regulates late blight resistance and abiotic tolerance in potato and may have a potential use in engineering late blight resistance in potato.

The StDREB1 transcription factor is involved in oxidative stress response and enhances tolerance to salt stress

We have shown previously that the potato dehydration responsive element binding (StDREB1) transcription factor plays an important role in regulating and improving salt and drought stress-response genes in potato plant. To further characterize StDREB1 involvement in stress response, we focuse here on the investigation of the StDREB1 target genes by an Electrophoretic mobility shift assay. The data obtains indicated that the StDREB1 protein can bind to both GCC and DRE boxes in the promoter sequence of target genes, suggesting that this transcription factor may play a key role in the response to abiotic- and biotic-stresses by the activation of the DRE- and GCC- mediated signaling pathways. In a second step, since some DREB factors were related to the oxidative stress response, we showed that H2O2 treatment led to a significant increase of StDREB1 expression in wild- type potato plants. Moreover, the analysis of the oxidative stress response of StDREB1 transgenic potato plants revealed lower levels of H2O2 and malondialdehyde than wild-type control plants submitted in vitro to salt stress. An increase in the antioxidant enzyme activities including superoxide dismutase (SOD) and catalase was also observed in StDREB1 plants. In addition, an enhanced expression of the Cu/Zn SOD gene was noticed in these StDREB1 transgenic plants, cultivated under salt stress conditions. These results suggest that StDREB1 plays an essential role in the regulation of stress-response by regulating the oxidative stress response. The involvement of this transcription factor in the activation of osmoprotectant synthesis was also confirmed.

Arabidopsis AtERF71/HRE2 functions as transcriptional activator via cis-acting GCC box or DRE/CRT element and is involved in root development through regulation of root cell expansion.

AtERF71/HRE2 binds to GCC box or DRE/CRT as transcription activator and plays an important role in root development via root cell expansion regulation. AtERF71/HRE2 transcription factor, a member of the AP2/ERF family, plays a key role in the stress response. GCC box and DRE/CRT, both essential cis-acting elements, have been shown to be recognized by AP2/ERF family transcription factors. However, it remains unclear whether or not AtERF71/HRE2 directly interacts with GCC box and/or DRE/CRT. Here, we showed that AtERF71/HRE2 binds to GCC box and DRE/CRT by electrophoretic mobility shift assay (EMSA). Binding of AtERF71/HRE2 to GCC box and DRE/CRT was also detected by fluorescence measurement and surface plasmon resonance spectroscopy (BIAcore) experiments. Folding properties of AtERF71/HRE2 proteins were characterized by CD spectroscopy, and AtERF71/HRE2 showed thermal stability as evidenced by two endothermic peaks (T d) at 53 and 65 °C. In addition, AtERF71/HRE2 showed transcriptional activation activity via GCC box and DRE/CRT in Arabidopsis protoplasts. Interestingly, AtERF71/HRE2 OXs showed increased primary root length due to elevated root cell expansion. Our data indicate that AtERF71/HRE2 binds to both GCC box and DRE/CRT, transactivates expression of genes downstream via GCC box or DRE/CRT, and plays an important role in root development through regulation of root cell expansion.

Isolation and characterization of ZmERF1 encoding ethylene responsive factor-like protein 1 in popcorn (Zea mays L.)

The ethylene response factor (ERF) plays a key role in the transcriptional regulation of plant growth, development, and response to various environmental perturbations. Study of ERF transcription factors, especially those in cultivated crops, could facilitate their molecular genetic modification. In this study, an ERF1 gene, designated as ZmERF1, which was found to be localized in the nucleus, was isolated from popcorn (Zea mays L.). Further analysis showed that the ZmERF1 proteins could bind to the GCC box motif and act as a transcriptional activator. A clear picture of ZmERF1 expression pattern showed that ZmERF1 expression varies during different development stages of endosperm. Furthermore, its expression could be highly induced by salinity, osmotic and heat stress, suggesting that the gene is involved in stress signaling pathways in maize. Therefore, ZmERF1 could play important role in abiotic stress tolerance and endosperm development in maize.

The CarERF genes in chickpea (Cicer arietinum L.) and the identification of CarERF116 as abiotic stress responsive transcription factor.

The AP2/ERF family is one of the largest transcription factor gene families that are involved in various plant processes, especially in response to biotic and abiotic stresses. Complete genome sequences of one of the world's most important pulse crops chickpea (Cicer arietinum L.), has provided an important opportunity to identify and characterize genome-wide ERF genes. In this study, we identified 120 putative ERF genes from chickpea. The genomic organization of the chickpea ERF genes suggested that the gene family might have been expanded through the segmental duplications. The 120 member ERF family was classified into eleven distinct groups (I-X and VI-L). Transcriptional factor CarERF116, which is differentially expressed between drought tolerant and susceptible chickpea cultivar under terminal drought stress has been identified and functionally characterized. The CarERF116 encodes a putative protein of 241 amino acids and classified into group IX of ERF family. An in vitro CarERF116 protein-DNA binding assay demonstrated that CarERF116 protein specifically interacts with GCC box. We demonstrate that CarERF116 is capable of transactivation activity of and show that the functional transcriptional domain lies at the C-terminal region of the CarERF116. In transgenic Arabidopsis plants overexpressing CarERF116, significant up-regulation of several stress related genes were observed. These plants also exhibit resistance to osmotic stress and reduced sensitivity to ABA during seed germination. Based on these findings, we conclude that CarERF116 is an abiotic stress responsive gene, which plays an important role in stress tolerance. In addition, the present study leads to genome-wide identification and evolutionary analyses of chickpea ERF gene family, which will facilitate further research on this important group of genes and provides valuable resources for comparative genomics among the grain legumes.

The Arabidopsis ethylene/jasmonic acid-NRT signaling module coordinates nitrate reallocation and the trade-off between growth and environmental adaptation.

Stresses decouple nitrate assimilation and photosynthesis through stress-initiated nitrate allocation to roots (SINAR), which is mediated by the nitrate transporters NRT1.8 and NRT1.5 and functions to promote stress tolerance. However, how SINAR communicates with the environment remains unknown. Here, we present biochemical and genetic evidence demonstrating that in Arabidopsis thaliana, ethylene (ET) and jasmonic acid (JA) affect the crosstalk between SINAR and the environment. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that ethylene response factors (ERFs), including OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59, bind to the GCC boxes in the NRT1.8 promoter region, while ETHYLENE INSENSITIVE3 (EIN3) binds to the EIN3 binding site motifs in the NRT1.5 promoter. Genetic assays showed that cadmium and sodium stresses initiated ET/JA signaling, which converged at EIN3/EIN3-Like1 (EIL1) to modulate ERF expression and hence to upregulate NRT1.8. By contrast, ET and JA signaling mediated the downregulation of NRT1.5 via EIN3/EIL1 and other, unknown component(s). SINAR enhanced stress tolerance and decreased plant growth under nonstressed conditions through the ET/JA-NRT1.5/NRT1.8 signaling module. Interestingly, when nitrate reductase was impaired, SINAR failed to affect either stress tolerance or plant growth. These data suggest that SINAR responds to environmental conditions through the ET/JA-NRT signaling module, which further modulates stress tolerance and plant growth in a nitrate reductase-dependent manner.

Arabidopsis HRE1α, a splicing variant of AtERF73/HRE1, functions as a nuclear transcription activator in hypoxia response and root development

HRE1α shows transcriptional activation activity in its C-terminal region via GCC box but not DRE/CRT and plays an important role in root development via root meristem cell division regulation. AtERF73/HRE1 protein, a member of the Arabidopsis AP2/ERF family, contains a conserved AP2/ERF DNA-binding domain. Here, we studied the molecular function of HRE1α, a splicing variant of AtERF73/HRE1, as well as its role in root development. HRE1α-overexpressing transgenic plants (OXs) showed tolerance to submergence. HRE1α showed transcriptional activation activity via GCC box but not DRE/CRT. The 121-211 aa region of HRE1α was responsible for the transcriptional activation activity, and the region was conserved among homologs of other species but was not found in other Arabidopsis proteins. HRE1α OXs showed increased primary root length due to elevated root cell division. Our results suggest that HRE1α functions as a transcription activator in the nucleus, and plays an important role in root development through regulation of root meristem cell division.

RsERF1 derived from wild radish (Raphanus sativus) confers salt stress tolerance in Arabidopsis

The change in environmental parameters affects normal growth of plants, eventually reduces agricultural production. Ethylene plays vital roles in plant stress responses, germination, fruit ripening, organ abscission, pathogen response, and senescence. Expression of an ethylene-responsive transcription factor (ERF) was induced in Korean halophyte, Raphanus sativus var. hortensis f. raphanistroides (wild radish) by 200-mM sodium chloride (NaCl). Raphanus sativus ethylene-responsive transcription factor 1 (RsERF1) is also localized to nucleus, similar to other transcription factors. In yeast, RsERF1 showed transcriptional activation property, by expressing the reporter gene. Being a TF, RsERF1 specifically bound to the cis-acting elements, GCC box and DRE/CRT in vitro, to initiate transcription. Homozygous T3 transgenic Arabidopsis, overexpressing RsERF1, showed significant tolerance against salt stress in soil-grown conditions. The tolerance was also marked by an increased germination rate of RsERF1 transgenics in salt-containing media. In RsERF1 overexpression lines, abiotic stress-related genes such as ABF3, ABF4, ADH, Rab18, and SUS1 were upregulated by 200-mM NaCl. ERFs have been studied and proven for their tolerance potential against various abiotic stresses, but RsERF1 belongs to an ERF subgroup called ethylene-responsive transcription factor related to AP2 (ERF-RAP2). Thus, this is a first report for ERF-RAP2 from Korean halophyte cDNA library. We believe that extensive posttranslational modification studies will reveal the role and location of RsERF1 in stress tolerance pathway.

The Wheat Ethylene Response Factor Transcription Factor PATHOGEN-INDUCED ERF1 Mediates Host Responses to Both the Necrotrophic Pathogen Rhizoctonia cerealis and Freezing Stresses

Sharp eyespot disease (primarily caused by the pathogen Rhizoctonia cerealis) and freezing stress are important yield limitations for the production of wheat (Triticum aestivum). Here, we report new insights into the function and underlying mechanisms of an ethylene response factor (ERF) in wheat, Pathogen-Induced ERF1 (TaPIE1), in host responses to R. cerealis and freezing stresses. TaPIE1-overexpressing transgenic wheat exhibited significantly enhanced resistance to both R. cerealis and freezing stresses, whereas TaPIE1-underexpressing wheat plants were more susceptible to both stresses relative to control plants. Following both stress treatments, electrolyte leakage and hydrogen peroxide content were significantly reduced, and both proline and soluble sugar contents were elevated in TaPIE1-overexpressing wheat, whereas these physiological traits in TaPIE1-underexpressing wheat exhibited the opposite trend. Microarray and quantitative reverse transcription-polymerase chain reaction analyses of TaPIE1-overexpressing and -underexpressing wheat plants indicated that TaPIE1 activated a subset of defense- and stress-related genes. Assays of DNA binding by electrophoretic mobility shift and transient expression in tobacco (Nicotiana tabacum) showed that the GCC boxes in the promoters of TaPIE1-activated genes were essential for transactivation by TaPIE1. The transactivation activity of TaPIE1 and the expression of TaPIE1-activated defense- and stress-related genes were significantly elevated following R. cerealis, freezing, and exogenous ethylene treatments. TaPIE1-mediated responses to R. cerealis and freezing were positively modulated by ethylene biosynthesis. These data suggest that TaPIE1 positively regulates the defense responses to R. cerealis and freezing stresses by activating defense- and stress-related genes downstream of the ethylene signaling pathway and by modulating related physiological traits in wheat.

The Arabidopsis transcriptional repressor ERF9 participates in resistance against necrotrophic fungi

Complex plant defenses that include the hypersensitive response (HR) are mediated by plant hormones, such as salicylic acid (SA), jasmonic acid (JA) and ethylene. We previously isolated the Arabidopsis DEAR1 (DREB AND EAR MOTIF PROTEIN 1) regulator and showed that its overexpression DEAR1 (DEAR1ox) resulted in a dwarf phenotype and lesion-like cell death, accompanied by elevated expression of PR (PATHOGENESIS-RELATED) genes. Here, we show that transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) has enhanced resistance to the necrotrophic fungus Botrytis cinerea (B. cinerea). This result indicates that DEAR1 represses negative regulators of plant defense responses, including transcriptional repressors belonging to the ERF (ETHYLEN RESPONSE FACTOR) family. Knockout mutants of ERF9 (erf9), which were down-regulated in DEAR1ox plants, showed transcriptional promotion of PDF1.2 (PATHOGEN-INDUCIBLE PLANT DEFENSIN) genes, which serve as positive markers for the ethylene/jasmonic acid (JA) signaling pathway and provide enhanced resistance to B. cinerea. Biochemical assays demonstrated that the ERF9 in capable of binding to the GCC box, a cis-element contained in the promoters of the PDF1.2 gene that possesses trans-repression activity. Moreover, infection with B. cinerea resulted in the promotion of the PDF1.2 expression, coinciding with suppression of the ERF9 gene under the control of the DEAR1 gene. These results indicate that the transcriptional repressor ERF9 participates in plant defense mechanisms against necrotic fungi mediated by the DEAR1-dependent ethylene/JA signaling pathway.

The Arabidopsis ETHYLENE RESPONSE FACTOR1 Regulates Abiotic Stress-Responsive Gene Expression by Binding to Different cis-Acting Elements in Response to Different Stress Signals

ETHYLENE RESPONSE FACTOR1 (ERF1) is an upstream component in both jasmonate (JA) and ethylene (ET) signaling and is involved in pathogen resistance. Accumulating evidence suggests that ERF1 might be related to the salt stress response through ethylene signaling. However, the specific role of ERF1 in abiotic stress and the molecular mechanism underlying the signaling cross talk still need to be elucidated. Here, we report that ERF1 was highly induced by high salinity and drought stress in Arabidopsis (Arabidopsis thaliana). The salt stress induction required both JA and ET signaling but was inhibited by abscisic acid. ERF1-overexpressing lines (35S:ERF1) were more tolerant to drought and salt stress. They also displayed constitutively smaller stomatal aperture and less transpirational water loss. Surprisingly, 35S:ERF1 also showed enhanced heat tolerance and up-regulation of heat tolerance genes compared with the wild type. Several suites of genes activated by JA, drought, salt, and heat were found in microarray analysis of 35S:ERF1. Chromatin immunoprecipitation assays found that ERF1 up-regulates specific suites of genes in response to different abiotic stresses by stress-specific binding to GCC or DRE/CRT. In response to biotic stress, ERF1 bound to GCC boxes but not DRE elements; conversely, under abiotic stress, we observed specific binding of ERF1 to DRE elements. Furthermore, ERF1 bound preferentially to only one among several GCC box or DRE/CRT elements in the promoter region of its target genes. ERF1 plays a positive role in salt, drought, and heat stress tolerance by stress-specific gene regulation, which integrates JA, ET, and abscisic acid signals.

Expression of signalling and defence‐related genes mediated by over‐expression of JERF1, and increased resistance to sheath blight in rice

Sheath blight (SB) is one of the most destructive rice diseases that affect the yield and quality of rice. Jasmonate and Ethylene Response Factor 1, designated JERF1, can be induced by ethylene, methyl jasmonate (MeJA), abscisic acid (ABA) and salt stress. Previous research demonstrated that over‐expression of JERF1 enhanced drought tolerance by regulating the expression of stress‐related genes in rice. The present study reports that JERF1 could improve SB resistance of transgenic rice. At the molecular and physiological levels, JERF1 could up‐regulate the expression of signalling and defence‐related genes and increase the activities of lipoxygenase (LOX) and phenylalanine ammonia lyase (PAL). Analysis of cis‐elements showed that there were several DRE/CRT (dehydration‐responsive element/C‐repeat) and/or GCC boxes in the promoters of these candidate genes, indicating that JERF1 might regulate the expression of defence‐related genes via interaction with these cis‐elements. These results suggest that JERF1 might improve SB resistance by modulating the expression of defence‐related genes. This may prove to be a useful strategy for genetic engineering to improve SB resistance using transcription factor genes in rice.

AaERF1 positively regulates the resistance to Botrytis cinerea in Artemisia annua.

Plants are sessile organisms, and they can not move away under abiotic or biotic stresses. Thus plants have evolved a set of genes that response to adverse environment to modulate gene expression. In this study, we characterized and functionally studied an ERF transcription factor from Artemisia annua, AaERF1, which plays an important role in biotic stress responses. The AaERF1 promoter had been cloned and GUS staining results of AaERF1 promoter-GUS transgenic A. annua showed that AaERF1 is expressed ubiquitiously in all organs. Several putative cis-acting elements such as W-box, TGA-box and Py-rich element, which are involved in defense responsiveness, are present in the promoter. The expression of AaERF1 can be induced vigorously by methyl jasmonate as well as by ethephon and wounding, implying that AaERF1 may activate some of the defense genes via the jasmonic acid and ethylene signaling pathways of A. annua. The results of electrophoretic mobility shift assay (EMSA) and yeast one-hybrid experiments showed that AaERF1 was able to bind to the GCC box cis-acting element in vitro and in yeast. Ectopic expression of AaERF1 could enhance the expression levels of the defense marker genes PLANT DEFENSIN1.2 (PDF1.2) and BASIC CHITINASE (ChiB), and increase the resistance to Botrytis cinerea in the 35S::AaERF1 transgenic Arabidopsis. The down-regulated expression level of AaERF1 evidently reduced the resistance to B. cinerea in A. annua. The overall results showed that AaERF1 positively regulated the resistance to B. cinerea in A. annua.

Expression Profile in Rice Panicle: Insights into Heat Response Mechanism at Reproductive Stage

Rice at reproductive stage is more sensitive to environmental changes, and little is known about the mechanism of heat response in rice panicle. Here, using rice microarray, we provided a time course gene expression profile of rice panicle at anther developmental stage 8 after 40°C treatment for 0 min, 20 min, 60 min, 2 h, 4 h, and 8 h. The identified differentially expressed genes were mainly involved in transcriptional regulation, transport, cellular homeostasis, and stress response. The predominant transcription factor gene families responsive to heat stress were Hsf, NAC, AP2/ERF, WRKY, MYB, and C2H2. KMC analysis discovered the time-dependent gene expression pattern under heat stress. The motif co-occurrence analysis on the promoters of genes from an early up-regulated cluster showed the important roles of GCC box, HSE, ABRE, and CE3 in response to heat stress. The regulation model central to ROS combined with transcriptome and ROS quantification data in rice panicle indicated the great importance to maintain ROS balance and the existence of wide cross-talk in heat response. The present study increased our understanding of the heat response in rice panicle and provided good candidate genes for crop improvement.