Haiwen Zhang

Transcriptional Modulation of Ethylene Response Factor Protein JERF3 in the Oxidative Stress Response Enhances Tolerance of Tobacco Seedlings to Salt, Drought, and Freezing

Abiotic stresses such as drought, cold, and salinity affect normal growth and development in plants. The production and accumulation of reactive oxygen species (ROS) cause oxidative stress under these abiotic conditions. Recent research has elucidated the significant role of ethylene response factor (ERF) proteins in plant adaptation to abiotic stresses. Our earlier functional analysis of an ERF protein, JERF3, indicated that JERF3-expressing tobacco (Nicotiana tabacum) adapts better to salinity in vitro. This article extends that study by showing that transcriptional regulation of JERF3 in the oxidative stress response modulates the increased tolerance to abiotic stresses. First, we confirm that JERF3-expressing tobacco enhances adaptation to drought, freezing, and osmotic stress during germination and seedling development. Then we demonstrate that JERF3-expressing tobacco imparts not only higher expression of osmotic stress genes compared to wild-type tobacco, but also the activation of photosynthetic carbon assimilation/metabolism and oxidative genes. More importantly, this regulation of the expression of oxidative genes subsequently enhances the activities of superoxide dismutase but reduces the content of ROS in tobacco under drought, cold, salt, and abscisic acid treatments. This indicates that JERF3 also modulates the abiotic stress response via the regulation of the oxidative stress response. Further assays indicate that JERF3 activates the expression of reporter genes driven by the osmotic-responsive GCC box, DRE, and CE1 and by oxidative-responsive as-1 in transient assays, suggesting the transcriptional activation of JERF3 in the expression of genes involved in response to oxidative and osmotic stress. Our results therefore establish that JERF3 activates the expression of such genes through transcription, resulting in decreased accumulation of ROS and, in turn, enhanced adaptation to drought, freezing, and salt in tobacco.

Overexpression of an ERF transcription factor TSRF1 improves rice drought tolerance

One of the major limitations in rice production is a shortage of water. Conventional breeding as well as emerging genetic engineering methods may be used to improve plant stress tolerance. Some transcription factors regulating stress responsive genes have become important target genes for improving plant drought tolerance. Previously, we have shown that a tomato ethylene response factor (ERF) protein TSRF1 that binds to GCC box in the promoters of pathogenesis-related genes positively regulates pathogen resistance in tomato and tobacco, but negatively regulates osmotic response in tobacco. Here, we further report the ability of TSRF1 to regulate osmotic and drought responses in monocot rice. TSRF1 improves the osmotic and drought tolerance of rice seedlings without growth retardation, as determined by physiological analyses of root and leaf growth, leaf water loss and survival rate under stress. In addition, the amounts of proline and soluble sugars in transgenic rice lines increase by 30%-60% compared to those in wild-type plants. Moreover, TSRF1 activates the expression of a putative rice abscisic acid (ABA) synthesis gene SDR, resulting in enhanced ABA sensitivity in transgenic rice. TSRF1 also increases the expression of MYB, MYC and proline synthesis and photosynthesis-related genes, probably by binding to dehydration responsive element and GCC boxes in promoters of the target genes. These results demonstrate that TSRF1 enhances the osmotic and drought tolerance of rice by modulating the increase in stress responsive gene expression.

Transcriptional regulation of the ethylene response factor LeERF2 in the expression of ethylene biosynthesis genes controls ethylene production in tomato and tobacco.

Fine-tuning of ethylene production plays an important role in developmental processes and in plant responses to stress, but very little is known about the regulation of ethylene response factor (ERF) proteins in ethylene biosynthesis genes and ethylene production. Identifying cis-acting elements and transcription factors that play a role in this process, therefore, is important. Previously, a tomato (Solanum lycopersicum [f. sp. Lycopersicon esculentum]) ERF protein, LeERF2, an allele of TERF2, was reported to confer ethylene triple response on plants. This paper reports the transcriptional modulation of LeERF2/TERF2 in ethylene biosynthesis in tomato and tobacco (Nicotiana tabacum). Using overexpressing and antisense LeERF2/TERF2 transgenic tomato, we found that LeERF2/TERF2 is an important regulator in the expression of ethylene biosynthesis genes and the production of ethylene. Expression analysis revealed that LeERF2/TERF2 is ethylene inducible, and ethylene production stimulated by ethylene was suppressed in antisense LeERF2/TERF2 transgenic tomato, indicating LeERF2/TERF2 to be a positive regulator in the feedback loop of ethylene induction. Further research showed that LeERF2/TERF2 conservatively modulates ethylene biosynthesis in tobacco and that such regulation in tobacco is associated with the elongation of the hypocotyl and insensitivity to abscisic acid and glucose during germination and seedling development. The effects on ethylene synthesis were similar to those of another ERF protein, TERF1, because TERF1 and LeERF2/TERF2 have overlapping roles in the transcriptional regulation of ethylene biosynthesis in tobacco. Biochemical analysis showed that LeERF2/TERF2 interacted with GCC box in the promoter of NtACS3 and with dehydration-responsive element in the promoter of LeACO3, resulting in transcriptional activation of the genes for ethylene biosynthesis in tomato and tobacco, which is a novel regulatory function of ERF proteins in plant ethylene biosynthesis.

Expression of ethylene response factor JERF1 in rice improves tolerance to drought.

Ethylene response factor (ERF) proteins regulate a variety of stress responses in plant. JERF1, a tomato ERF protein, can be induced by abscisic acid (ABA). Overexpression of JERF1 enhanced the tolerance of transgenic tobacco to high salt concentration, osmotic stress, and low temperature by regulating the expression of stress-responsive genes by binding to DRE/CRT and GCC-box cis-elements. In this research, we further report that overexpression of JERF1 significantly enhanced drought tolerance of transgenic rice. The overexpression activated the expression of stress-responsive genes and increased the synthesis of the osmolyte proline by regulating the expression of OsP5CS, encoding the proline biosynthesis key enzyme deltal-pyrroline-5-carboxylate synthetase. JERF1 also activated the expression of two ABA biosynthesis key enzyme genes, OsABA2 and Os03g0810800, and increased the synthesis of ABA in rice. Analysis of cis-elements of JERF1-targeted genes pointed to the existence of DRE/CRT and/or GCC box in their promoters, indicating that JERF1 could activate the expression of related genes in rice by binding to these cis-elements. Unlike some other ERF proteins, constructive overexpression of JERF1 did not change the growth and development of transgenic rice, which makes JEFR1 a potentially useful source in breeding for greater tolerance to abiotic stress.

The ethylene response factor AtERF11 that is transcriptionally modulated by the bZIP transcription factor HY5 is a crucial repressor for ethylene biosynthesis in Arabidopsis.

The phytohormones abscisic acid (ABA) and ethylene are known to play multiple roles in plant development and stress responses. Ethylene biosynthesis is affected by several factors including drought, cold and the phytohormone auxin, although the role of ABA is unclear. In this work ABA-responsive mutants were screened and a bZIP transcription factor HY5 was identified as a negative regulator of ethylene biosynthesis via modulation of the expression of the ethylene biosynthesis genes ACS2 and ACS5. Members of the ethylene response factor (ERF) family of transcriptional repressors in Arabidopsis have been shown to modulate ABA responses and three ERF members were found to carry putative HY5-binding cis-acting elements. Analyses with biochemical and molecular approaches revealed that HY5 specifically binds to the G-box region of the AtERF11 promoter to activate its transcription. We further demonstrate that AtERF11, which contains a repressor motif at its C-terminal, interacts with the dehydration-responsive element in the ACS2/5 promoters, to repress its expression, resulting in decreased ethylene biosynthesis. Moreover, an AtERF11 knockout mutant showed increased levels of ACS2/5 expression and ethylene emission, while treatment with ABA greatly suppressed ACS5 transcripts but not ACS2 expression and the ethylene content, indicating that AtERF11 is a key negative regulator for ABA-mediated control of ethylene synthesis. In addition, in ethylene over-producer mutants, ABA treatment was shown to suppress ACS5 transcripts and ethylene content, thereby affecting growth and development. Based on these data, in this research we present a model suggesting that the HY5-AtERF11 regulon is a key factor modulating ABA-regulated ethylene biosynthesis.

Expression of TERF1 in rice regulates expression of stress-responsive genes and enhances tolerance to drought and high-salinity

Drought and high-salinity are the important constraints that severely affect plant development and crop yield worldwide. It has been established that ethylene response factor (ERF) proteins play important regulatory roles in plant response to abiotic and biotic stresses. Our previous researches have revealed that transgenic tobacco over-expressing TERF1 (encoding a tomato ERF protein) showed enhanced tolerance to abiotic stress. Here, we further investigate the function of TERF1 in transgenic rice. Compared with the wild-type plants, overexpression of TERF1 resulted in an increased tolerance to drought and high-salt in transgenic rice. And the enhanced tolerance may be associated with the accumulation of proline and the decrease of water loss. Furthermore, TERF1 can effectively regulate the expression of stress-related functional genes Lip5, Wcor413-l, OsPrx and OsABA2, as well as regulatory genes OsCDPK7, OsCDPK13 and OsCDPK19 under normal growth conditions. Our analyses of cis-acting elements show that there exist DRE/CRT and/or GCC-box existing in TERF1 targeted gene promoters. Our results revealed that ectopic expression of TERF1 in rice caused a series of molecular and physiological alterations and resulted in the transgenic rice with enhanced tolerance to abiotic stress, indicating that TERF1 might have similar regulatory roles in response to abiotic stress in tobacco and rice.

ERF protein JERF1 that transcriptionally modulates the expression of abscisic acid biosynthesis-related gene enhances the tolerance under salinity and cold in tobacco

Increasing evidences indicate that ethylene responsive factor (ERF) proteins regulate a variety of biotic and abiotic stress responses, and plant development as well. Previously we demonstrated that JERF1, encoding an ERF transcriptional activator, is inducible by ethylene, MeJA, ABA, and NaCl, suggesting its possible regulation in multiple stress responses. In the present paper, we report that expressing JERF1 in tobacco increases the seed germination under mannitol treatment, and enhances the tolerance to high salinity and low temperature, through accumulating sodium in vacuole of leaves and stabilizing the plasma membrane, respectively, and significantly increases the growth of tobacco roots and leaves under salinity and low temperature through an unknown mechanism. The evidence that JERF1 interacts with multiple cis-acting elements, such as GCC-box, DRE, and CE1, to activate the expression of stress-related genes, supports the possible involvement of JERF1 in multiple plant stress responses with ABA-dependent and ABA-independent manner. More importantly, we reveal that expressing JERF1 in tobacco transcriptionally regulates the expression of ABA biosynthesis-related gene NtSDR, resulting in the increase of the ABA content. Together, our results indicate that JERF1 interacts with multiple cis-acting elements and activates the expression of stress responsive and ABA biosynthesis-related genes, consequently causing ABA biosynthesis, and ultimately enhancing tobacco tolerance and growth under high salinity and low temperature.

An ethylene response factor OsWR1 responsive to drought stress transcriptionally activates wax synthesis related genes and increases wax production in rice

Increasing evidence has revealed the major enzymes-involved in Arabidopsis and maize wax/cutin synthesis; however, there is limited information about the genes-associated with wax/cutin synthesis in rice. Here we report the characterization of an ethylene response factor gene in rice. This rice wax synthesis regulatory gene 1 (OsWR1) is a homolog of Arabidopsis wax/cutin synthesis regulatory gene WIN1/SHN1. Transcript analysis showed that OsWR1 is induced by drought, abscisic acid and salt, and is predominantly expressed in leaves. Functional analyses indicated that overexpressing OsWR1 (Ox-WR1) improved while RNA interference OsWR1 rice (RI-WR1) decreased drought tolerance, consistent with water loss and cuticular permeability, suggesting that OsWR1-triggered drought response might be associated with cuticular characteristics. In addition, OsWR1 activated the expression of the genes-related to oxidative stress response and membrane stability. Gas chromatograph–mass spectrometry analysis further showed that OsWR1 modulated the wax synthesis through alteration of long chain fatty acids and alkanes, evidencing the regulation of OsWR1 in wax synthesis. Detection with real-time PCR amplification indicated that Ox-WR1 enhanced while RI-WR1 decreased the expression of wax/cutin synthesis related genes. Furthermore, OsWR1 physically interacted with the DRE and GCC box in the promoters of wax related genes OsLACS2 and OsFAE1’-L, indicating that OsWR1 at least directly modulates the expression of these genes. Thus our results indicate that OsWR1 is a positive regulator of wax synthesis related genes in rice, and this regulation, distinct from its homology regulator of WIN1/SHN1 in cutin synthesis, subsequently contributes to reduced water loss and enhanced drought tolerance.

Functional analyses of ethylene response factor JERF3 with the aim of improving tolerance to drought and osmotic stress in transgenic rice

Ethylene response factor (ERF) proteins play important roles in regulating plant stress response and development. Our previous studies have shown that JERF3 activates the expression of oxidative stress responsive genes in transgenic tobacco and enhances tolerance to salt, drought, and freezing, indicating that JERF3 is a very important transcriptional regulator in dicot plants. In the study reported here, we further addressed the regulatory function of JERF3 in a monocot, rice, by generating transgenic rice plants overexpressing JERF3 and comparing these with non-transgenic rice plants for physiological and molecular alterations and tolerance to drought and osmotic stresses. Growth and development under normal growth conditions were the same in both the transgenic and non-transgenic rice. Interestingly, the JERF3 transgenic plants exhibited better stress tolerance, whereas the non-transgenic rice seedlings showed serious stress symptoms and ultimately died after the drought and osmotic treatments. Biochemical analysis revealed that the contents of soluble sugars and proline were significantly increased in transgenic rice compared with non-transgenic plants under dehydration conditions. In addition, overexpression of JERF3 in rice led to the up-regulated expression of two OsP5CS genes in response to drought treatment compared with their expression in non-transgenic plants. JERF3 also activated the expression of stress-responsive genes, including WCOR413-like, OsEnol, and OsSPDS2, in transgenic rice under normal growth conditions. These data suggest that JERF3 plays important roles in transgenic rice and that it is likely to be beneficial in engineering crop plants with improved tolerance to drought and osmotic stresses.

Transcriptional activation of OsDERF1 in OsERF3 and OsAP2-39 negatively modulates ethylene synthesis and drought tolerance in rice.

The phytohormone ethylene is a key signaling molecule that regulates a variety of developmental processes and stress responses in plants. Transcriptional modulation is a pivotal process controlling ethylene synthesis, which further triggers the expression of stress-related genes and plant adaptation to stresses; however, it is unclear how this process is transcriptionally modulated in rice. In the present research, we report the transcriptional regulation of a novel rice ethylene response factor (ERF) in ethylene synthesis and drought tolerance. Through analysis of transcriptional data, one of the drought-responsive ERF genes, OsDERF1, was identified for its activation in response to drought, ethylene and abscisic acid. Transgenic plants overexpressing OsDERF1 (OE) led to reduced tolerance to drought stress in rice at seedling stage, while knockdown of OsDERF1 (RI) expression conferred enhanced tolerance at seedling and tillering stages. This regulation was supported by negative modulation in osmotic adjustment response. To elucidate the molecular basis of drought tolerance, we identified the target genes of OsDERF1 using the Affymetrix GeneChip, including the activation of cluster stress-related negative regulators such as ERF repressors. Biochemical and molecular approaches showed that OsDERF1 at least directly interacted with the GCC box in the promoters of ERF repressors OsERF3 and OsAP2-39. Further investigations showed that OE seedlings had reduced expression (while RI lines showed enhanced expression) of ethylene synthesis genes, thereby resulting in changes in ethylene production. Moreover, overexpression of OsERF3/OsAP2-39 suppressed ethylene synthesis. In addition, application of ACC recovered the drought-sensitive phenotype in the lines overexpressing OsERF3, showing that ethylene production contributed to drought response in rice. Thus our data reveal that a novel ERF transcriptional cascade modulates drought response through controlling the ethylene synthesis, deepening our understanding of the regulation of ERF proteins in ethylene related drought response.