No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
The Arabidopsis AP2/ERF transcription factor RAP2.6 participates in ABA, salt and osmotic stress responses
Abstract
AP2/ERF proteins play crucial roles in various biological processes. RAP2.6, an Arabidopsis AP2/ERF family member, has been reported to function in plant response to biotic stress, but whether it also functions in plant response to abiotic stress is not known. In this work, we demonstrate that in wild-type Arabidopsis, the expression of RAP2.6 is responsive to abscisic acid (ABA) and different stress conditions such as high salt, osmotic stress, and cold. Trans-activating ability tests in yeast demonstrate that RAP2.6 could act as a transactivator. RAP2.6 is able to bind to the GCC and CE1 cis-elements, as confirmed by both electrophoretic mobility shift assay (EMSA) and yeast one-hybrid assay. Experiments with RAP2.6-YFP fusion protein indicated that RAP2.6 is nuclear localized. Overexpression of RAP2.6 conferred hypersensitivity to exogenous ABA and abiotic stresses during seed germination and early seedling growth in Arabidopsis. The ABA content in RAP2.6 overexpressor lines decreased after being treated with salt. Furthermore, transcripts of AtABI4 and some stress inducible genes increased, and loss of ABI4 function rescues the hypersensitive phenotype of RAP2.6 overexpression lines under ABA and stress treatment. These results suggest that RAP2.6 participates in abiotic stress, possibly through the ABA-dependent pathway.
... RAP2.6, an important transcription factor belonging to ERF subfamily (Guo et al., 2005), could be activated by C-repeat binding factor (CBF) (Fowler and Thomashow, 2002). RAP2.6 play a key role in response to ABA, JA, wounding, cold and drought stresses (Chen et al., 2002;He et al., 2004;Zhu et al. 2010Zhu et al. , 2020. Krishnaswamy et al. (2008Krishnaswamy et al. ( , 2011 found that the expression of RAP2.6 in salt-stressed Arabidopsis was significantly higher than that in unstressed Arabidopsis. ...
... It is very interesting that rap2.6 mutant was insensitive to salt stress although RAP2.6 has played an important role in many abiotic stresses such as wounding, cold, drought and osmotic stresses (Chen et al., 2002;He et al., 2004;Zhu et al. 2010Zhu et al. , 2020. Based on these results, we proposed two possibilities that RAP2.6 is the negative regulator to salt stress in Arabidopsis seedlings or the absence of RAP2.6 could lead to a loss of plant response to salt stress. ...
The transcription factors of the AP2/ERF family are involved in plant growth and development and responses to biotic and abiotic stresses. Here, we found RAP2.6, a transcription factor which belongs to the ERF subfamily, was responsive to salt stress in Arabidopsis. Under salt stress conditions, rap2.6 mutant seedlings were the sensitivity deficiency to salt stress which was reflected in higher germination rate and longer root length compared to the wild type. Also, the expressions of salt-related gene including SOS1, SOS2, SOS3, NHX1, NHX3, NHX5 and HKT1 in rap2.6 mutant seedlings were lower than the wild type under salt stress. rap2.6 mutant adult lacked salt stress tolerance based on the results of the phenotype, survival rates and ion leakage. Compared to wild type, rap2.6 mutant adult accumulated more Na+ in leaves and roots while the salt-related gene expressions were lower. In addition, the photosynthetic electron transport and PSII energy distribution in rap2.6 mutant plant leaves had been more seriously affected under salt stress conditions compared to the wild type. In summary, this study identified essential roles of RAP2.6 in regulating salt stress tolerance in Arabidopsis.
... RAP2.6, similar to ERF96, an Arabidopsis AP2/ERF family member, has also been found to function in the plant response to salt, drought, cold, ABA stresses and so on (Ali et al. 2013). RAP2.6 can bind to CE1 and the GCC-box, which are important responsive elements in abscisic acid (ABA) and ethylene signaling (Zhu et al. 2010). There were also few studies about the interaction between RAP2.6 and other related genes. ...
... HMT1 and HMT2 were induced by high Se as expected (Fig.S3b, Fig. 4c), and the transcription levels of HMT2 were significantly higher in OE-1 and OE-6 plants than in WT and rap2.6 plants (Fig. 4c), but HMT3 expression level were lower in plants grown on 30 µM Na 2 SeO 3 medium than those grown on ½ MS medium for all plant lines (Fig.S3c). This may be because AtHMT2 has the highest homology to AbSMT plant response to biotic stress (Ali et al. 2013) and abiotic stress (Zhu et al. 2010). In this study, we reveal that RAP2.6 positively regulates Se stress response in Arabidopsis thaliana through genetic evidence. ...
Selenium (Se) in right amount is beneficial for plant growth and adaptation to environmental condition, but the mechanism of high Se stress in plants is unknown. Ethylene response factors (ERFs) is one of the most important families that are involved in plant response to biotic and abiotic stresses. Here, we provide evidence for a role of RAP2.6, encoding a member of the ERF subfamily B-4 of AP2/ERF transcription factor family, in selenite tolerance in Arabidopsis. RAP2.6 transcripts were rapidly increased in response to 30 µM selenite in the first 3 h, suggesting it roles in the high Se stress. Overexpression of RAP2.6 actually improved Arabidopsis plants resistance to Se stress, while rap2.6 mutant and wild type (WT) plants demonstrated similar resistance to selenite stress. Moreover, under Se stress conditions, the Se content of overexpression plants was significantly lower than that of WT and mutants. Further study indicated that overexpression of RAP2.6 reduced transcript levels of phosphate transporters PHT1;4. Meanwhile, overexpression of RAP2.6 increased transcription levels of chloroplastic Nifs-like cysteine desulfurase (CpNifS), homocysteine methyltransferase (HMT) and S-adenosyl-L-methionine: L-methionine S-methyltransferase (MMT), thus reduced the nonspecific incorporation of selenocysteine (SeCys) into proteins. In addition, the antioxidant activity in the RAP2.6-overexpressing plants had clearly elevated. Under selenite stress, overexpression of RAP2.6 significantly increased catalase (CAT) and ascorbate peroxidase (APX) activities and glutathione (GSH) content, with a decreased level of reactive oxygen species (ROS) compared to WT. In summary, the results indicated that RAP2.6 plays a positive role in regulating selenium tolerance in Arabidopsis thaliana.
... In A. thaliana, AtRAP2.6 has been recognized as an important regulator of plant meristem regeneration and exerts a vital role in recovering cell damage. Moreover, the activity of the RAP2.6 gene was inversely proportional to the concentration of auxin IAA, highlighting its inhibitory role in IAA synthesis [58]. Combined with the expression changes of the MnAP2/ERF gene family in five tissues, we found that most MnAP2/ERFs have some tissue expression specificity. ...
The AP2/ERF gene family, referring to an exclusive class of transcription factors unique to plants, is involved in various biological processes, including plant growth and responses to environmental stresses like high salt and drought. In this study, the AP2/ERF gene in M. notabilis was comprehensively identified and bioinformatically analyzed based on the genomic data of M. notabilis. 106 members in the MnAP2/ERF gene family were identified in the M. notabilis genome and were categorized into five subfamilies: ERF, AP2, DREB, RAV, and Soloist, with the ERF subfamily representing 80.19% of the total. The MnAP2/ERF gene family was observed to be distributed on six chromosomes of M. notabilis. Members in the MnAP2/ERF gene family exhibited obvious differences in amino acid number, molecular weight, isoelectric point, and other properties. Approximately 68.87% of the MnAP2/ERF proteins were acidic, all exhibiting hydrophilic characteristics. Differences in conserved sequences and arrangement of AP2 domains were observed among distinct subfamilies, with genes in the same subfamily sharing similar conserved domain compositions. There were 47 genes without untranslated regions and 44 genes with two untranslated regions. The upstream functions of promoters were concentrated on light reactions and plant hormones. Evolutionarily, significant structural differences were identified, and 28 MnAP2/ERF gene family proteins could interact with each other. Moreover, 35 family genes were involved in 22 fragment repeat events, and 55 MnAP2/ERF and 84 AtAP2/ERF genes showed collinearity. The expression of the MnAP2/ERF gene family was significantly different in different parts, indicating that these gene family members were involved in different physiological activities. These results established a theoretical foundation for investigating the functional and evolutionary aspects of AP2/ERF gene family genes in M. notabilis, as well as exploring the root morphogenesis of M. notabilis. Additionally, this study contributes to a basis for the improvement of cultivar stress resistance of M. notabilis.
... Chr2B_Cda09636 encodes the ethylene-responsive transcription factor (AP2/ERF) protein, which is suggested to regulate salt stress tolerance in plants via an ABA-dependent system. Overexpression of its homologous gene RAP2.6, for example, imparted hypersensitivity to exogenous ABA and abiotic stresses, whereas rap2.6 mutants were salt stress insensitive and accumulated more Na+ in leaves and stems (Zhu et al., 2010(Zhu et al., , 2020. The bermudagrass Chr2B_Cda09636 gene is likely to perform functions in controlling cellular ion homeostasis, which impacts salt tolerance. ...
Bermudagrass ( Cynodon dactylon ) is a globally distributed, extensively used warm‐season turf and forage grass with high tolerance to salinity and drought stress in alkaline environments. However, the origin of the species and genetic mechanisms for salinity tolerance in the species are basically unknown. Accordingly, we set out to study evolution divergence events in the Cynodon genome and to identify genes for salinity tolerance. We developed a 604.0 Mb chromosome‐level polyploid genome sequence for bermudagrass ‘A12359’ ( n = 18). The C. dactylon genome comprises 2 complete sets of homoeologous chromosomes, each with approximately 30 000 genes, and most genes are conserved as syntenic pairs. Phylogenetic study showed that the initial Cynodon species diverged from Oropetium thomaeum approximately 19.7–25.4 million years ago (Mya), the A and B subgenomes of C. dactylon diverged approximately 6.3–9.1 Mya, and the bermudagrass polyploidization event occurred 1.5 Mya on the African continent. Moreover, we identified 82 candidate genes associated with seven agronomic traits using a genome‐wide association study, and three single‐nucleotide polymorphisms were strongly associated with three salt resistance genes: RAP2‐2 , CNG channels, and F14D7.1 . These genes may be associated with enhanced bermudagrass salt tolerance. These bermudagrass genomic resources, when integrated, may provide fundamental insights into evolution of diploid and tetraploid genomes and enhance the efficacy of comparative genomics in studying salt tolerance in Cynodon .
... Among these 17 genes, 4 were involved in the ABA-dependent drought stress response. RAP2-6 has been identified as an ABA-dependent abiotic response gene; the overexpression of RAP2-6 enhances drought and salt stress resistance in transgenic plants [41,42]. MSL3 is an osmotic stress-response gene; msl2 msl3 mutants express proline and ABA metabolism genes under drought or osmotic stress [43]. ...
Drought stress is seriously affecting the growth and production of crops, especially when agricultural irrigation still remains quantitatively restricted in some arid and semi-arid areas. The identification of drought-tolerant genes is important for improving the adaptability of maize under stress. Here, we found that a new member of the actin-depolymerizing factor (ADF) family; the ZmADF5 gene was tightly linked with a consensus drought-tolerant quantitative trait locus, and the significantly associated signals were detected through genome wide association analysis. ZmADF5 expression could be induced by osmotic stress and the application of exogenous abscisic acid. Its overexpression in Arabidopsis and maize helped plants to keep a higher survival rate after water-deficit stress, which reduced the stomatal aperture and the water-loss rate, as well as improved clearance of reactive oxygen species. Moreover, seventeen differentially expressed genes were identified as regulated by both drought stress and ZmADF5, four of which were involved in the ABA-dependent drought stress response. ZmADF5-overexpressing plants were also identified as sensitive to ABA during the seed germination and seedling stages. These results suggested that ZmADF5 played an important role in the response to drought stress.
... Regulatory genes, particularly transcription factors, are gaining attention. Many transcription factors associated with cold tolerance, such as AP2/ EREBP, WRKY, MYB, bZIP, TCP, NAC, and Zinc-finger (Riechmann et al., 2000;Zhu et al., 2010;Iwase et al., 2011), have been identified. However, only MYB, bZIP, and TCP transcription factors have been identified in chrysanthemums so far, indicating the need for further research to discover other transcription factors. ...
Chrysanthemums are one of the top ten most well-known traditional famous flowers in China and one of the top four cut flowers worldwide, holding a significant position in landscape gardening. The cold temperatures of winter restrict the cultivation, introduction, and application of chrysanthemum, resulting in high costs for year-round production. This severely impacts the ornamental and economic value of chrysanthemum. Therefore, research on cold tolerance is of vital importance for guiding chrysanthemum production and application. With the development of genomics, transcriptomics, metabolomics, and other omics approaches, along with high-throughput molecular marker technologies, research on chrysanthemum cold tolerance has been continuously advancing. This article provides a comprehensive overview of the progress in cold tolerance research from various aspects, including chrysanthemum phenotype, physiological mechanisms, the forward genetics, molecular mechanisms, and breeding. The aim is to offer insights into the mechanisms of cold tolerance in chrysanthemum and provide reference for in-depth research and the development of new cold tolerance chrysanthemum varieties.
... The AP2/ERF TF family contains four major subfamilies in plants, including AP2, RAV, ERF, and DREB. Many AP2/ERF genes have been reported to be involved in responses to a variety of environmental stimuli in plants [51,52]. Here, we identified eight DEGs encoding AP2, ERF, and SREB subfamily TFs, and four genes (Os01g07120/OsDREB2A, Os02g43790/AP59, Os04g32620/OsERF10, and Os05g27930/OsDREB2B) have been reported to be involved in the drought tolerance of rice [53][54][55][56]. ...
High temperature is one of the most important environmental factors influencing rice growth, development, and yield. Therefore, it is important to understand how rice plants cope with high temperatures. Herein, the heat tolerances of T2 (Jinxibai) and T21 (Taizhongxianxuan2hao) were evaluated at 45 ℃, and T21 was found to be sensitive to heat stress at the seedling stage. Analysis of the H2O2 and proline content revealed that the accumulation rate of H2O2 was higher in T21, whereas the accumulation rate of proline was higher in T2 after heat treatment. Meanwhile, transcriptome analysis revealed that several pathways participated in the heat response, including “protein processing in endoplasmic reticulum”, “plant hormone signal transduction”, and “carbon metabolism”. Additionally, our study also revealed that different pathways participate in heat stress responses upon prolonged stress. The pathway of “protein processing in endoplasmic reticulum” plays an important role in stress responses. We found that most genes involved in this pathway were upregulated and peaked at 0.5 or 1 h after heat treatment. Moreover, sixty transcription factors, including the members of the AP2/ERF, NAC, HSF, WRKY, and C2H2 families, were found to participate in the heat stress response. Many of them have also been reported to be involved in biotic or abiotic stresses. In addition, through PPI (protein–protein interactions) analysis, 22 genes were identified as key genes in the response to heat stress. This study improves our understanding of thermotolerance mechanisms in rice, and also lays a foundation for breeding thermotolerant cultivars via molecular breeding.
... By binding to the specific cis-acting elements in the promoters of the target genes, transcription factors (TFs) were considered as the most important regulators involved in the regulation of a broad range of target genes associated with plant tolerance and adaptation to environmental stress (Erpen et al. 2018;Li et al. 2019;Zhao et al. 2020;Zhu et al. 2010Zhu et al. , 2018. The expression pattern of the same TF family may differ in different plants under different abiotic stresses (Sun et al. 2023). ...
Key message
Multiple regulatory pathways of T. chinensis to salt stress were identified through transcriptome data analysis.
Abstract
Tamarix chinensis (Tamarix chinensis Lour.) is a typical halophyte capable of completing its life cycle in soils with medium to high salinity. However, the mechanisms underlying its resistance to high salt stress are still largely unclear. In this study, transcriptome profiling analyses in different organs of T. chinensis plants in response to salt stress were carried out. A total number of 2280, 689, and 489 differentially expressed genes (DEGs) were, respectively, identified in roots, stems, and leaves, with more DEGs detected in roots than in stems and leaves. Gene Ontology (GO) term analysis revealed that they were significantly enriched in “biological processes” and “molecular functions”. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that “Beta-alanine metabolism” was the most differentially enriched pathway in roots, stems, and leaves. In pair-to-pair comparison of the most differentially enriched pathways, a total of 14 pathways, including 5 pathways in roots and leaves, 6 pathways in roots and stems, and 3 pathways in leaves and stems, were identified. Furthermore, genes encoding transcription factor, such as bHLH, bZIP, HD-Zip, MYB, NAC, WRKY, and genes associated with oxidative stress, starch and sucrose metabolism, and ion homeostasis, were differentially expressed with distinct organ specificity in roots, stems, and leaves. Our findings in this research provide a novel approach for exploring the salt tolerance mechanism of halophytes and identifying new gene targets for the genetic breeding of new plant cultivars with improved resistance to salt stress.
... The CREs containing four times GCCelements (AGCCGCC), four times CE1-elements (TGCCACCGG) and four times DRE elements (TACCGACAT) was inserted into pLacZi vector via SmaI and XhoI sites. The vectors were introduced into yeast strain EGY48 through LiAc-mediated transformation method (Clontech, Shanghai, China grown on the Trp-and Ura-deficient medium supplemented with 0.2 mM X-gal were used for color change observation (Zhu et al., 2010). Yeast strain EGY48 expressing empty pB42AD and pLacZi vector was used as the negative control. ...
Dehydration response element binding (DREB) proteins are vital for plant abiotic stress responses, but the understanding of DREBs in bamboo, an important sustainable non-timber forest product, is limited. Here we conducted a comprehensive genome-wide analysis of the DREB gene family in Moso bamboo, representing the most important running bamboo species in Asia. In total, 44 PeDREBs were identified, and information on their gene structures, protein motifs, phylogenetic relationships, and stress-related cis-regulatory elements (CREs) was provided. Based on the bioinformatical analysis, we further analyzed PeDREBs from the A5 group and found that four of five PeDREB transcripts were induced by salt, drought, and cold stresses, and their proteins could bind to stress-related CREs. Among these, PeDREB28 was selected as a promising candidate for further functional characterization. PeDREB28 is localized in nucleus, has transcriptional activation activity, and could bind to the DRE- and coupling element 1- (CE1) CREs. Overexpression of PeDREB28 in Arabidopsis and bamboo improved plant abiotic stress tolerance. Transcriptomic analysis showed that broad changes due to the overexpression of PeDREB28. Furthermore, 628 genes that may act as the direct PeDREB28 downstream genes were identified by combining DAP-seq and RNA-seq analysis. Moreover, we confirmed that PeDREB28 could bind to the promoter of pyrabactin-resistance-like gene (DlaPYL3), which is a homolog of abscisic acid receptor in Arabidopsis, and activates its expression. In summary, our study provides important insights into the DREB gene family in Moso bamboo, and contributes to their functional verification and genetic engineering applications in the future.
... The AtERF53, AtRAP2.6L and AtRAP2. 6 can be induced by ABA [20][21][22]. And OsERF71 was demonstrated to positively modulate ABA signaling pathway [23]. ...
Background
The 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.
Result
In 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.
Conclusions
Overall, 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.
... In addition, ERF transcription factors can also bind to CT-rich (T(T/A)ACC GCC TT), JERE (JA and elicitor responsive element) and AS-1 (activation sequence-1 element) sequences. Research shows that RAP2.6 can bind to the GCC box and CE1 cis-elements, respond to different stress conditions, such as high salt, cold, and osmotic stress, and participate in salt and osmotic stress responses (Zhu et al. 2010). ORA47 (octadecanoid responsive AP2/ERF domain transcription factor 47) has been shown to bind to the cis-element (NC/GT)CGNCCA, which plays a role in the biosynthesis of JA and ABA signaling pathways to regulate the biosynthesis of a suite of phytohormone genes when plants are subjected to wounding and water stress (Chen et al. 2016). ...
Plants usually encounter multifactorial stresses in which two or more abiotic and/or biotic stress factors are present simultaneously. Under multifactorial stress, plant growth and survival are severely reduced, even if the levels of each individual stress are very low. Therefore, discovering multifactorial stress tolerance mechanisms is important for coordinating plant growth and uncovering plant response to combinations of two or more stress conditions. AP2/ERF (APETALA2/ethylene responsive factor) transcription factors (TFs) affect different hormone signaling pathways to regulate diverse developmental processes and stress responses. AP2/ERF TFs have specific protein and gene interaction partners that participate in the regulation of the stress response. AP2/ERF TFs are involved in the regulation of gene expression by specifically binding to cis elements in the promoter region of target genes. In this review, we have summarized the mechanism of AP2/ERF TFs in multifactorial stress responses. We have reviewed the recent major developments regarding the regulation and function of these genes, highlighting their role in multifactorial stress tolerance in plants.
... AtERF12 involved in the regulation of seed dormancy downstream of ETR1 and promoting floral meristem identity (Li et al., 2019;Chandler and Werr, 2020). Overexpression of RAP2.6 conferred hypersensitivity to exogenous ABA and abiotic stresses during seed germination and early seedling growth in Arabidopsis (Zhu et al., 2010). In this study, we observed that several AP2/ERFs, including ZoAP2-3 and ZoERF17, the homologs of AIL5 and AtERF12 respectively, displayed significantly differential expression between young and matured rhizomes. ...
Ginger (Zingiber officinale Roscoe) is an important vegetable with medicinal value. Rhizome development determines ginger yield and quality. However, little information is available about the molecular features underlying rhizome expansion and maturation. In this study, we investigated anatomy characteristics, lignin accumulation and transcriptome profiles during rhizome development. In young rhizomes, the vascular bundle (VB) was generated with only vessels in it, whereas in matured rhizomes, three to five layers of fibre bundle in the xylem were formed, resulting in VB enlargement. It indicates VB development favouring rhizome swelling. With rhizome matured, the lignin content was remarkably elevated, thus facilitating tissue lignification. To explore the regulators for rhizome development, nine libraries including ginger young rhizomes (GYR), growing rhizomes (GGR), and matured rhizomes (GMR) were established for RNA-Seq, a total of 1264 transcription factors (TFs) were identified. Among them, 35, 116, and 14 differentially expressed TFs were obtained between GYR and GGR, GYR and GMR, and GGR and GMR, respectively. These TFs were further divided into three categories. Among them, three ZobHLHs (homologs of Arabidopsis LHW and AtbHLH096) as well as one DIVARICATA homolog in ginger might play crucial roles in controlling VB development. Four ZoWRKYs and two ZoNACs might be potential regulators associated with rhizome maturation. Three ZoAP2/ERFs and one ZoARF might participate in rhizome development via hormone signalling. This result provides a molecular basis for rhizome expansion and maturation in ginger.
... We also find binding sites for RAP2.6, ABI3VP1/VRN1 and DEAR4, involved in ABA, 400 osmotic, drought and salt stress responses (Wang et al., 2020a;Wang et al., 2020b;Zhang et al., 2020;Zhu et al., 2010b), 401 for RRTF1 and RAP2.3, which mediate plant defense responses against Botrytis cinerea and other pathogens (León et 402 al., 2020;Wang et al., 2020a), for ORA47, a regulator of general stress responses induced by methyl jasmonate (Zeng et 403 al., 2022), and for DREB2C, AP2EREBP and G2like_tnt.At3g13040, involved in response to drought and dehydration 404 (Dietz et al., 2010;Kizis et al., 2001;Lee et al., 2010;Riechmann and Meyerowitz, 1998;Wang et al., 2022), corroborating the NAC61 role in abiotic and biotic stress responses (Supplementary Fig. S6). ...
During late-and post-ripening stages, grape berry undergoes profound biochemical and physiological changes whose molecular control is poorly understood. Here, we report the role of NAC61, a grapevine NAC transcription factor, in regulating different processes featuring the berry ripening progression.
NAC61 is highly expressed during post-harvest berry dehydration and its expression pattern is closely related to sugar concentration. The ectopic expression of NAC61 in Nicotiana benthamiana leaves determines low stomatal conductance, high leaf temperature, tissue collapse and a higher relative water content. Transcriptome analysis of grapevine leaves transiently overexpressing NAC61, and DNA affinity purification and sequencing analyses allowed us to narrow down a list of NAC61-regulated genes. Direct regulation of the stilbene synthase regulator MYB14 , the osmotic stress-related gene DHN1b , the Botrytis cinerea susceptibility gene WRKY52 and the NAC61 itself, is validated. We also demonstrate that NAC61 interacts with NAC60, a proposed master regulator of grapevine organ maturation, in the activation of MYB14 and NAC61 expression. Overall, our findings establish NAC61 as a key player in a regulative network that governs stilbenoid metabolism and osmotic, oxidative and biotic stress responses in grape berry during late-and post-ripening.
Highlights
NAC61 regulates stilbene biosynthesis and abiotic/biotic stress responses that hallmark late-and post-ripening developmental stages in grapevine berry. NAC61 participates in a NAC60-dependent regulatory network, also triggering its self-activation.
... Zhu et al. found that overexpression of RAP2.6, a member of the AP2/ERF family, could induce hypersensitivity responses to exogenous ABA and abiotic stress during seed germination and early seedling growth of Arabidopsis. ABA content in RAP2.6 overexpressing lines decreased after salt treatment (Zhu et al., 2010). The AtRAP2.6L homologous apple gene MdERF113 positively regulates abiotic stress and ABA stress (Rui et al., 2023). ...
The ethylene response factor (ERF) transcription factors, which is one of the largest transcription factor families in plants, are involved in biological and abiotic stress response and play an important role in plant growth and development. In this study, the GmABR1 gene from the soybean inbred line Zhonghuang24 (ZH24)×Huaxia 3 (HX3) was investigated its aluminum (Al) tolerance. GmABR1 protein has a conserved domain AP2, which is located in the nucleus and has transcriptional activation ability. The results of real-time quantitative PCR (qRT-PCR) showed that the GmABR1 gene presented a constitutive expression pattern rich in the root tip, stem and leaf tissues of HX3. After Al stress, the GmABR1 transcript was significantly increased in the roots. The transcripts of GmABR1 in the roots of HX3 treated with 50 µM AlCl3 was 51 times than that of the control. The GmABR1 was spatiotemporally specific with the highest expression levels when Al concentration was 50 µM, which was about 36 times than that of the control. The results of hematoxylin staining showed that the root tips of GmABR1-overexpression lines were stained the lightest, followed by the control, and the root tips of GmABR1 RNAi lines were stained the darkest. The concentrations of Al3+ in root tips were 207.40 µg/g, 147.74 µg/g and 330.65 µg/g in wild type (WT), overexpressed lines and RNAi lines, respectively. When AlCl3 (pH4.5) concentration was 100 µM, all the roots of Arabidopsis were significantly inhibited. The taproot elongation of WT, GmABR1 transgenic lines was 69.6%, 85.6%, respectively. When treated with Al, the content of malondialdehyde (MDA) in leaves of WT increased to 3.03 µg/g, while that of transgenic Arabidopsis increased from 1.66-2.21 µg/g, which was lower than that of WT. Under the Al stress, the Al stress responsive genes such as AtALMT1 and AtMATE, and the genes related to ABA pathway such as AtABI1, AtRD22 and AtRD29A were up-regulated. The results indicated that GmABR1 may jointly regulate plant resistance to Al stress through genes related to Al stress response and ABA response pathways.
... Plant-specific AP2/ERF transcription factors can regulate plant responses to environmental stimuli or plant growth and development, depending on the presence of a highly conserved 60-amino-acid AP2 domain in the protein [38,39]. For example, rice OsEREBP1 attenuates disease caused by Xanthomonas and confers drought and submergence tolerance by activating the jasmonate and abscisic acid signaling pathways, thereby priming the rice plants for enhanced survival under abiotic or biotic stress conditions [40]. A. thaliana RAP2.6 participates in abiotic stress, including abscisic acid (ABA), salt and osmotic stress, possibly through the ABA-dependent pathway [41]. A previous study found that StAP2/ERFs are indispensable in Cd uptake and tolerance and may be useful in designing gene-modified potato plants with improved Cd tolerance [31]. ...
Cadmium (Cd) stress has a major impact on ecosystems, so it is important to find suitable Cd-tolerant plants while elucidating the responsible molecular mechanism for phytoremediation to manage Cd soil contamination. Iris lactea var. chinensis is an ornamental perennial groundcover plant with strong tolerance to Cd. Previous studies found that IlAP2, an AP2/ERF superfamily gene, may be an interacting partner of the metallothionein gene IlMT2a, which plays a key role in Cd tolerance. To study the role of IlAP2 in regulating Cd tolerance in I. lactea, we analyzed its regulation function and mechanism based on a yeast two-hybrid assay, a bimolecular fluorescence complementation test, quantitative real-time PCR, transgenics and transcriptome sequencing. The results showed that IlAP2 interacts with IlMT2a and may cooperate with other transcription factors to regulate genes involved in signal transduction and plant hormones, leading to reduced Cd toxicity by hindering Cd transport. These findings provide insights into the mechanism of IlAP2-mediated stress responses to Cd and important gene resources for improving plant stress tolerance in phytoremediation.
... It has been established that COR genes are induced by the ABAdependent and ABA-independent signaling pathways in A. thaliana (Fig. 7); with the ABA-independent pathway regulating cold stress through the CBF/DREB1. However, it was subsequently found that the ABA-dependent pathways are not completely independent, and there is an interactive process between the two pathways (Zhu et al., 2010). Previous studies have confirmed that ABA also can activate CRT promoters cis-acting elements. ...
... These results suggests that this set of genes may be useful for future evaluation of seed viability in A. fraxinifolium. Despite the antagonistic effects of signaling pathways of ethylene and ABA, the presence of another down-regulated gene, Ethylene-responsive transcription factor RAP2-12, suggests a complex interaction, since both inhibit root growth after germination [43][44][45]. Interestingly, among genes common to both treatments, casein kinase 1-like protein 1 was suggested as a positive mediator of ABA signaling in Arabidopsis [46], but in this study it was shown to be down-regulated. ...
Astronium fraxinifolium Schott (Anacardiaceae), also known as a ‘gonçalo-alves’, is a tree of the American tropics, with distribution in Mexico, part of Central America, Argentina, Bolivia, Brazil and Paraguay. In Brazil it is an endangered species that occurs in the Cerrado, Caatinga and in the Amazon biomes. In support of ex situ conservation, this work aimed to study two accessions with different longevity (p50) of A. fraxinifolium collected from two different geographic regions, and to evaluate the transcriptome during aging of the seeds in order to identify genes related to seed longevity. Artificial ageing was performed at a constant temperature of 45 °C and 60% relative humidity. RNA was extracted from 100 embryonic axes exposed to control and aging conditions for 21 days. The transcriptome analysis revealed differentially expressed genes such as Late Embryogenesis Abundant (LEA) genes, genes involved in the photosystem, glycine rich protein (GRP) genes, and several transcription factors associated with embryo development and ubiquitin-conjugating enzymes. Thus, these results contribute to understanding which genes play a role in seed ageing, and may serve as a basis for future functional characterization of the seed aging process in A. fraxinifolium.
... After ethephon treatment, most AP2/ERFs were downregulated in both flesh and receptacles . Moreover, ABA, auxin, MeJA, and brassinolide (BR) also mediate changes in plant growth and development through AP2/ERFs (Hu et al., 2004;Zhu et al., 2010;Gao et al., 2019;Lin et al., 2020). In turn, AP2/ERFs affect plant hormone synthesis. ...
Fig fruits have significant health value and are culturally important. Under suitable climatic conditions, fig fruits undergo a superfast ripening process, nearly doubling in size, weight, and sugar content over three days in parallel with a sharp decrease in firmness. In this study, 119 FcAP2/ERF genes were identified in the fig genome, namely 95 ERF s, 20 AP2 s, three RAV s, and one soloist . Most of the ERF subfamily members (76) contained no introns, whereas the majority of the AP2 subfamily members had at least two introns each. Three previously published transcriptome datasets were mined to discover expression patterns, encompassing the fruit peel and flesh of the ‘Purple Peel’ cultivar at six developmental stages; the fruit receptacle and flesh of the ‘Brown Turkey’ cultivar after ethephon treatment; and the receptacle and flesh of parthenocarpic and pollinated fruits of the ‘Brown Turkey’ cultivar. Eighty-three FcAP2/ERF s (68 ERF s, 13 AP2 s, one RAV , and one soloist ) were expressed in the combined transcriptome dataset. Most FcAP2/ERF s were significantly downregulated (|log 2 (fold change) | ≥ 1 and p -adjust < 0.05) during both normal fruit development and ethephon-induced accelerated ripening, suggesting a repressive role of these genes in fruit ripening. Five significantly downregulated ERFs also had repression domains in the C-terminal. Seven FcAP2/ERF s were identified as differentially expressed during ripening in all three transcriptome datasets. These genes were strong candidates for future functional genetic studies to elucidate the major FcAP2/ERF regulators of the superfast fig fruit ripening process.
... A substantial increase in the expression of ESR1 in WIND1 overexpressing lines and immunoprecipitation assays have shown that WIND1 effectively binds to ESR1 promoter thus upregulating its expression (Iwase et al., 2017). The RAP2.6 (At1g43160) (Zhu et al., 2010Xie et al., 2019) gene showed in Arabidopsis a similar expression pattern to WIND1, in terms of time after explant wounding and responsiveness to exogenous hormones, as it increases its expression when explants are transferred from high auxin-callus inducing medium to high-cytokinin shoot inducing medium (Iwase et al., 2017). Moreover, it is the closest homolog to two transcription factors characterized in tobacco that fulfills the same function as WIND1 (Iwase et al., 2017). ...
In vitro plant regeneration is a pivotal process in genetic engineering to obtain large numbers of transgenic, cisgenic and gene edited plants in the frame of functional gene or genetic improvement studies. However, several issues emerge as regeneration is not universally possible across the plant kingdom and many variables must be considered. In grapevine (Vitis spp.), as in other woody and fruit tree species, the regeneration process is impaired by a recalcitrance that depends on numerous factors such as genotype and explant-dependent responses. This is one of the major obstacles in developing gene editing approaches and functional genome studies in grapevine and it is therefore crucial to understand how to achieve efficient regeneration across different genotypes. Further issues that emerge in regeneration need to be addressed, such as somaclonal mutations which do not allow the regeneration of individuals identical to the original mother plant, an essential factor for commercial use of the improved grapevines obtained through the New Breeding Techniques. Over the years, the evolution of protocols to achieve plant regeneration has relied mainly on optimizing protocols for genotypes of interest whilst nowadays with new genomic data available there is an emerging opportunity to have a clearer picture of its molecular regulation. The goal of this review is to discuss the latest information available about different aspects of grapevine in vitro regeneration, to address the main factors that can impair the efficiency of the plant regeneration process and cause post-regeneration problems and to propose strategies for investigating and solving them.
... There have been several studies on transcription factors (TFs) from various families being activated in stress-response pathways, which are crucial for integrating salt sensory pathways and tolerance responses [13,21]. Genes belonging to the transcription factor family are differentially expressed in response to elevated external salinity including basic leucine zipper (bZIP) [22], WRKY [23], APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) [24], MYB [25], basic helix-loophelix (bHLH) [26], and NAC [27] families. The presence of NAC proteins (plant-specific TFs) confers salt stress resistance in plants [13,28,29]. ...
Mutations in the Betaine aldehyde dehydrogenase 2 (OsBadh2) gene resulted in aroma, which is a highly preferred grain quality attribute in rice. However, research on naturally occurring aromatic rice has revealed ambiguity and controversy regarding aroma emission, stress tolerance, and response to salinity. In this study, mutant lines of two non-aromatic varieties, Huaidao#5 (WT_HD) and Jiahua#1 (WT_JH), were generated by targeted mutagenesis of OsBadh2 using CRISPR/Cas9 technology. The mutant lines of both varieties became aromatic; however, WT_HD mutants exhibited an improved tolerance, while those of WT_JH showed a reduced tolerance to salt stress. To gain insight into the molecular mechanism leading to the opposite effects, comparative analyses of the physiological activities and expressions of aroma- and salinity-related genes were investigated. The WT_HD mutants had a lower mean increment rate of malondialdehyde, superoxide dismutase, glutamate, and proline content, with a higher mean increment rate of γ-aminobutyric acid, hydrogen peroxide, and catalase than the WT_JH mutants. Fluctuations were also detected in the salinity-related gene expression. Thus, the response mechanism of OsBadh2 mutants is complicated where the genetic makeup of the rice variety and interactions of several genes are involved, which requires more in-depth research to explore the possibility of producing highly tolerant aromatic rice genotypes.
... MYC2 was reported to modulate JA-dependent functions in Arabidopsis [85], for example, the expression of PDF1.2 that showed the same expression pattern as MYC2 in our experiment (Table 4) [86,87]. RAP2.6 and Rap2.6L were shown to be responsive to JA, SA, abscisic acid, and ethylene in addition to salt and drought [88][89][90]. WRKY75, though suggested to play a role in plant defense mechanisms, was shown to be mainly a modulator of phosphate acquisition [91]. Because of the striking representation of transcription factors modulating JA-dependent reactions, we concluded that some of the JA-related responses after Pst infection are delayed in Col-0 compared to C24. ...
Accessions of one plant species may show significantly different levels of susceptibility to stresses. The Arabidopsis thaliana accessions Col-0 and C24 differ significantly in their resistance to the pathogen Pseudomonas syringae pv. tomato (Pst). To help unravel the underlying mechanisms contributing to this naturally occurring variance in resistance to Pst, we analyzed changes in transcripts and compounds from primary and secondary metabolism of Col-0 and C24 at different time points after infection with Pst. Our results show that the differences in the resistance of Col-0 and C24 mainly involve mechanisms of salicylic-acid-dependent systemic acquired resistance, while responses of jasmonic-acid-dependent mechanisms are shared between the two accessions. In addition, arginine metabolism and differential activity of the biosynthesis pathways of aliphatic glucosinolates and indole glucosinolates may also contribute to the resistance. Thus, this study highlights the difference in the defense response strategies utilized by different genotypes.
... AP2/ERF superfamily transcription factors are one of the largest plant-specific transcriptional regulator groups in plants, with a conserved AP2/ERF DNA-binding structural domain of 57-66 amino acids in size (Okamuro et al., 1997). Ethylene responsive factors (ERFs) belong to AP2/ERF superfamily, which participate in plant response to hormone and abiotic stress (Qiang et al., 2010;Gibbs et al., 2015;Jung et al., 2017). In rice, overexpression of JERF3 and OsERF115/AP2/EREBP110 can increase the soluble sugar and proline content of transgenic plants, up-regulated the expression of P5CS gene under drought stress, and improve the tolerance of crops to drought and osmotic stress (Thoenes et al., 2004;Zhang and Huang, 2010). ...
Medicago falcata L. is an important legume forage grass with strong drought resistant, which could be utilized as an important gene pool in molecular breed of forage grass. In this study, M. falcata seedlings were treated with 400 mM mannitol to simulate drought stress, and the morphological and physiological changes were investigated, as well as the transcriptome changes of M. falcata seedlings at different treatment time points (0 h, 2 h, 6 h, 12 h, 24 h, 36 h and 48 h). Transcriptome analyses revealed four modules were closely related with drought response in M. falcata by WGCNA analysis, and four ERF transcription factor genes related with drought stress were identified ( MfERF053 , MfERF9 , MfERF034 and MfRAP2.1 ). Among them, MfERF053 was highly expressed in roots, and MfERF053 protein showed transcriptional activation activity by transient expression in tobacco leaves. Overexpression of MfERF053 in Arabidopsis improved root growth, number of lateral roots and fresh weight under drought, salt stress and exogenous ABA treatments. Transgenic Arabidopsis over-expressing MfERF053 gene grew significantly better than the wild type under both drought stress and salt stress when grown in soil. Taken together, our strategy with transcriptome combined WGCNA analyses identified key transcription factor genes from M. falcata , and the selected MfERF053 gene was verified to be able to enhance drought and salt resistance when over-expressed in Arabidopsis .
... ABA is another important phytohormone related to plant stress that mediates the plant responses to environmental factors, such as temperature, nitrate, and water stress (Lee and Luan, 2012;Hong et al., 2013). These environmental factors can affect plant growth, especially that of the root structure, and promote the growth of roots in one area while inhibiting the growth of roots in another area (Zhu et al., 2010). Many studies have reported that ABA contributes greatly to root development in model plant species, and ectopic expression of OBP4 or loss of RSL2 function in Arabidopsis thaliana has been shown to lead to ABA-insensitive root hair growth, confirming that OBP4-mediated RSL2 transcriptional inhibition contributes to ABA-dependent inhibition of root hair growth (Rymen et al., 2017). ...
The root system is essential for the stable growth of plants. Roots help anchor plants in the soil and play a crucial role in water uptake, mineral nutrient absorption and endogenous phytohormone formation. Root-restriction (RR) cultivation, a powerful technique, confines plant roots to a specific soil space. In the present study, roots of one-year-old “Muscat Hamburg” grapevine under RR and control (nR) treatments harvested at 70 and 125 days after planting were used for transcriptome sequencing, and in total, 2031 (nR7 vs. nR12), 1445 (RR7 vs. RR12), 1532 (nR7 vs. RR7), and 2799 (nR12 vs. RR12) differentially expressed genes (DEGs) were identified. Gene Ontology (GO) enrichment analysis demonstrated that there were several genes involved in the response to different phytohormones, including abscisic acid (ABA), auxin (IAA), ethylene (ETH), gibberellins (GAs), and cytokinins (CTKs). Among them, multiple genes, such as PIN2 and ERF113, are involved in regulating vital plant movements by various phytohormone pathways. Moreover, following RR cultivation, DEGs were enriched in the biological processes of plant-type secondary cell wall biosynthesis, the defense response, programmed cell death involved in cell development, and the oxalate metabolic process. Furthermore, through a combined analysis of the transcriptome and previously published microRNA (miRNA) sequencing results, we found that multiple differentially expressed miRNAs (DEMs) and DEG combinations in different comparison groups exhibited opposite trends, indicating that the expression levels of miRNAs and their target genes were negatively correlated. Furthermore, RR treatment indeed significantly increased the ABA content at 125 days after planting and significantly decreased the IAA content at 70 days after planting. Under RR cultivation, most ABA biosynthesis-related genes were upregulated, while most IAA biosynthesis-related genes were downregulated. These findings lay a solid foundation for further establishing the network through which miRNAs regulate grapevine root development through target genes and for further exploring the molecular mechanism through which endogenous ABA and IAA regulate root architecture development in grapevine.
... Both CDK8 and SnRK2.6 interact with RAP2.6, an ERF/AP2 transcription factor that is a substrate of SnRK2.6 in ABA signaling and response [94]. RAP2.6 directly binds to the promoters of ABA-responsive genes such as RD29A and COLD-REGULATED 15A (COR15A) with GCC or DRE elements and promotes their expression [94,95] (Figure 4). In cdk8 mutants, importantly, ABA-induced expression of both RAP2.6 and RAP2.6-regulated ...
As sessile organisms, plants are constantly exposed to a variety of environmental stresses and have evolved adaptive mechanisms, including transcriptional reprogramming, in order to survive or acclimate under adverse conditions. Over the past several decades, a large number of gene-specific transcription factors have been identified in the transcriptional regulation of plant adaptive responses. The Mediator complex plays a key role in transducing signals from gene-specific transcription factors to the transcription machinery to activate or repress target gene expression. Since its first purification about 15 years ago, plant Mediator complex has been extensively analyzed for its composition and biological functions. Mutants of many plant Mediator subunits are not lethal but are compromised in growth, development and response to biotic and abiotic stress, underscoring a particularly important role in plant adaptive responses. Plant Mediator subunits also interact with partners other than transcription factors and components of the transcription machinery, indicating the complexity of the regulation of gene expression by plant Mediator complex. Here, we present a comprehensive discussion of recent analyses of the structure and function of plant Mediator complex, with a particular focus on its roles in plant adaptive responses to a wide spectrum of environmental stresses and associated biological processes.
... Osmotic stress-responsive gene expression is regulated by ABA-dependent and ABA-independent pathways [41]. In the ABA-dependent pathway, numerous types of TFs, such as MYeloBlastosis (MYB), a basic helix-loop-helix (bHLH), the basic region leucine zipper (bZIP), ethylene response factor (ERF) and homeodomain TF are involved [42]. It was proven that overexpression of JERF1 (ERF gene) significantly enhanced drought tolerance of transgenic rice [43]. ...
Cereal plants under abiotic or biotic stressors to survive unfavourable conditions and continue growth and development, rapidly and precisely identify external stimuli and activate complex molecular, biochemical, and physiological responses. To elicit a response to the stress factors, interactions between reactive oxygen and nitrogen species, calcium ions, mitogen-activated protein kinases, calcium-dependent protein kinases, calcineurin B-like interacting protein kinase, phytohormones and transcription factors occur. The integration of all these elements enables the change of gene expression, and the release of the antioxidant defence and protein repair systems. There are still numerous gaps in knowledge on these subjects in the literature caused by the multitude of signalling cascade components, simultaneous activation of multiple pathways and the intersection of their individual elements in response to both single and multiple stresses. Here, signal transduction pathways in cereal plants under drought, salinity, heavy metal stress, pathogen, and pest attack, as well as the crosstalk between the reactions during double stress responses are discussed. This article is a summary of the latest discoveries on signal transduction pathways and it integrates the available information to better outline the whole research problem for future research challenges as well as for the creative breeding of stress-tolerant cultivars of cereals.
... The AP2/ERF TFs family is one of the largest plant-specific TFs families that share a wellconserved DNA-binding domain [80][81][82]. A lot of studies have shown genes belonging to AP2/ERF type TFs can increase tolerances to drought [83,84]. In this study, both the total number and up-regulation rate of genes belonging to the AP2/ERF TFs family increased with the extension of drought time, and DT was higher than DS (except DT21) at the same time point, which is in line with a previous study on tobacco [85]. ...
Drought is one of the most serious abiotic stress factors limiting crop yields. Although sunflower is considered a moderate drought-tolerant plant, drought stress still has a negative impact on sunflower yield as cultivation expands into arid regions. The extent of drought stress is varieties and time-dependent, however, the molecular response mechanisms of drought tolerance in sunflower with different varieties are still unclear. Here, we performed comparative physiological and transcriptome analyses on two sunflower inbred lines with different drought tolerance at the seedling stage. The analysis of nine physiological and biochemical indicators showed that the leaf surface area, leaf relative water content, and cell membrane integrity of drought tolerance inbred line were higher than those of drought-sensitive inbred line under drought stress, indicating that DT had stronger drought resistance. Transcriptome analyses identified 24,234 differentially expressed genes (DEGs). Gene ontology (GO) analysis showed the up-regulated genes were mainly enriched in gibberellin metabolism and rRNA processing, while the down-regulated genes were mainly enriched in cell-wall, photosynthesis, and terpene metabolism. Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis showed genes related to GABAergic synapse, ribosome biogenesis were up-regulated, while genes related with amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, photosynthesis were down-regulated. Mapman analysis revealed differences in plant hormone-signaling genes over time and between samples. A total of 1,311 unique putative transcription factors (TFs) were identified from all DEGs by iTAK, among which the high abundance of transcription factor families include bHLH, AP2/ERF, MYB, C2H2, etc. Weighted gene co-expression network analysis (WGCNA) revealed a total of 2,251 genes belonging to two modules(blue 4, lightslateblue), respectively, which were significantly associated with six traits. GO and KEGG enrichment analysis of these genes was performed, followed by visualization with Cytoscape software, and the top 20 Hub genes were screened using the CytoHubba plugin.
... DEHYDRATION RESPONSIVE ELEMENT-BINDING PROTEIN2A (DREB2A), DREB AND EAR MOTIF PROTEIN4 (DEAR4), and RELATED TO AP2.6 (RAP2.6) function as positive or negative regulators of salt stress tolerance in Arabidopsis thaliana (Kasuga et al., 1999;Zhu et al., 2010;Zhang et al., 2020). In addition, AP2/ERF family genes in rice (Oryza sativa; OsDREB1D and SALT-RESPONSIVE ERF1 [SERF1]), pea (Pisum sativum; PsAP2), and wheat (Triticum aestivum; TaERF3 and TaERF4) have been reported to confer salt stress tolerance (Dong et al., 2012;Rong et al., 2014;Schmidt et al., 2013;Mishra et al., 2015). ...
Leaf senescence proceeds with age but is modulated by various environmental
stresses and hormones. Salt stress is one of the most well-known environmental
stresses that accelerate leaf senescence. However, the molecular mechanisms that
integrate salt stress signaling with leaf senescence programs remain elusive. In this
study, we characterized the role of ETHYLENE RESPONSIVE FACTOR34 (ERF34),
an Arabidopsis APETALA2 (AP2)/ERF family transcription factor, in leaf senescence.
ERF34 was differentially expressed under various leaf senescence-inducing
conditions, and negatively regulated leaf senescence induced by age, dark, and salt
stress. ERF34 also promoted salt stress tolerance at different stages of the plant life
cycle such as seed germination and vegetative growth. Transcriptome analysis
revealed that the overexpression of ERF34 increased the transcript levels of salt
stress-responsive genes including COLD-REGULATED15A (COR15A), EARLY
RESPONSIVE TO DEHYDRATION10 (ERD10), and RESPONSIVE TO
DESICCATION29A (RD29A). Moreover, ERF34 directly bound to ERD10 and
RD29A promoters and activated their expression. Our findings indicate that ERF34
plays a key role in the convergence of the salt stress response with the leaf senescence
programs, and is a potential candidate for crop improvement, particularly by
enhancing salt stress tolerance.
Crop pests have profoundly deleterious effects on crop yield and food security. However, conventional pest control depends heavily on the utilization of insecticides, which develops strong pesticide resistance and concerns of food safety. Crop and their wild relatives display diverse levels of pest resistance, indicating the feasibility for breeding of pest-resistant crop varieties. In this study, we integrate deep learning (DL)/machine learning (ML) algorithms, plant phenomics and whole genome sequencing (WGS) data to conduct genomic selection (GS) of pest-resistance in grapevine. We employ deep convolutional neural networks (DCNN) to accurately calculate the severity of damage by pests on grape leaves, which achieves a classification accuracy of 95.3% (Visual Geometry Group 16, VGG16, for binary trait) and a correlation coefficient of 0.94 in regression analysis (DCNN with Pest Damage Score, DCNN-PDS, for continuous trait). We apply DL models to predict and integrate phenotype (both binary and continuous) along with WGS data from 231 grape accessions, conducting Genome-Wide Association Studies (GWAS). This analysis detects a total of 69 QTLs, encompassing 139 candidate genes involved in pathways associated with pest resistance, including jasmonic acid (JA), salicylic acid (SA), ethylene, and other related pathways. Furthermore, through the combination with transcriptome data, we identify specific pest-resistant genes, such as ACA12 and CRK3, which play distinct roles in resisting herbivore attacks. Machine learning-based GS demonstrates a high accuracy (95.7%) and a strong correlation (0.90) in predicting the leaf area damaged by pests as binary and continuous traits in grapevine, respectively. In general, our study highlights the power of DL/ML in plant phenomics and GS, facilitating genomic breeding of pest-resistant grapevine.
Perennial ryegrass ( Lolium perenne L.) is an outstanding turfgrass and forage cultivated in temperate regions worldwide. However, poor tolerance to extreme cold, heat, or drought limits wide extension and cultivation. DEHYDRATION‐RESPONSIVE ELEMENT BINDING FACTOR1s (DREB1s) play a vital role in enhancing plant tolerance to abiotic stress, specifically for low‐temperature stress. In this study, a total of 24 LpDREB1 family members were identified from the released genome of perennial ryegrass. Phylogenetic analysis showed that the LpDREB1 genes are divided into 7 groups that have close relationships with rice homologues. Conserved motif analysis revealed that members within the same group have similar conserved motif compositions. All LpDREB1s lack introns, and the promoter sequences of LpDREB1 genes contain multiple cis ‐acting elements associated with stress response, phytohormone signal transduction and plant growth and development. The majority of LpDREB1 genes were upregulated by drought, submergence, heat and cold stress treatments, including LpDREB1H2 . Further investigation showed that LpDREB1H2 is localized in the nucleus. Overexpression of LpDREB1H2 in Arabidopsis induced the expression of cold‐responsive (COR) genes, increased the levels of osmotic adjusting substances, and enhanced antioxidant enzyme activities, thus improving the cold tolerance of Arabidopsis. This study lays a foundation for further understanding the function of LpDREB1 genes in perennial ryegrass and provides insights for plant stress tolerance breeding.
During late- and post-ripening stages, grape berry undergoes profound biochemical and physiological changes whose molecular control is poorly understood. Here, we report the role of NAC61, a grapevine NAC transcription factor, in regulating different processes involved in berry ripening progression. NAC61 is highly expressed during post-harvest berry dehydration and its expression pattern is closely related to sugar concentration. The ectopic expression of NAC61 in Nicotiana benthamiana leaves resulted in low stomatal conductance, high leaf temperature, tissue collapse and a higher relative water content. Transcriptome analysis of grapevine leaves transiently overexpressing NAC61 and DNA affinity purification and sequencing analyses allowed us to narrow down a list of NAC61-regulated genes. Direct regulation of the stilbene synthase regulator MYB14, the osmotic stress-related gene DHN1b, the Botrytis cinerea susceptibility gene WRKY52, and NAC61 itself was validated. We also demonstrate that NAC61 interacts with NAC60, a proposed master regulator of grapevine organ maturation, in the activation of MYB14 and NAC61 expression. Overall, our findings establish NAC61 as a key player in a regulatory network that governs stilbenoid metabolism and osmotic, oxidative, and biotic stress responses that are the hallmark of late- and post-ripening grape stages.
The AP2/ERF TF (transcription factor) family is involved in regulating plant responses to various biotic and abiotic stresses. Nevertheless, understanding of the function of AP2/ERF TFs in wheat (Triticum aestivum L.) resistance against the obligate biotrophic stripe rust fungus (Puccinia striiformis f. sp tritici, Pst) remains limited. From a wheat-Pst incompatible interaction cDNA library, the transcript of TaAP2-10 was identified to be significantly induced during Pst infection. TaAP2-10, encodes an AP2 TF with two typical AP2-binding domains. There are three homologues of TaAP2-10 in the wheat genome, located on chromosome 6A, 6B and 6D. TaAP2-10 is localized in the nucleus of wheat protoplasts. A transactivation assay in yeast revealed that TaAP2-10 had transcriptional activation activity that was dependent on its C-terminal region. Quantitative real-time PCR (qRT-PCR) analyses verified that the expression of TaAP2-10 was specifically upregulated by avirulent Pst infection but not by virulent Pst, suggesting its role in wheat resistance to Pst. Furthermore, TaAP2-10 is also induced by abiotic stresses and hormone treatments, particularly under PEG4000 and abscisic acid (ABA) treatments, indicating its potential role in facilitating wheat adaptation to environmental stresses. Silencing TaAP2-10 by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) significantly reduced wheat resistance against Pst, resulting in a decreased reactive oxygen species (ROS) burst, and promoted Pst growth and development. These findings suggest that TaAP2-10, as a nuclear-localized transcription factor, positively regulates wheat resistance to Pst.
Several chemical substances are necessary for the growth and development of agricultural crop plants. Stress signals regulate an intricate network of transporters and metabolic activities in plants. Ca2+, nucleic acids, sugars and polyphosphoinositides, NO, ABA, jasmonic acids, salicylic acids, and polyamines are all examples of signal transduction molecules. A rise in Ca2+ levels is common when a crop plant is stressed because this is the typical secondary messenger in signal transduction pathways. Ca2+ sensors activate several receptors and pass them through the signaling chain to detect the presence of Ca2+ signals. Mitogen-activated protein kinases (MAPKs) may be triggered by hormones, hormone-like substances, environmental variables, and other stimuli. Phosphatidylinositols play an important role in intracellular signaling and plasma membranes. NO, a plant growth regulator, activates a variety of defense genes. Glycolysis, photosynthesis, nitrogen metabolism, such as reactive oxygen species and starch metabolization, defense systems, and cell cycle regulation are all dependent on sugar. ABA, JA, SA, and polyamines impact a wide range of stress responses. It is common for plants to respond against abiotic challenges through chemical signaling pathways. Crop plants are susceptible to abiotic stress, which may produce a wide range of signaling molecules. Abiotic stress response in agricultural crop plants is regulated by hormone signaling molecules, summarized in this chapter.
Inflictions caused by cold stress can result in disastrous effects on the productivity and survival of plants. Cold stress response in plants requires crosstalk between multiple signaling pathways including cold, heat, and reactive oxygen species (ROS) signaling networks. CBF, MYB, bHLH, and WRKY families are among the TFs that function as key players in the regulation of cold stress response at the molecular level. This review discusses some of the latest understanding on the regulation of expression and the mechanistic actions of plant TFs to address cold stress response. It was shown that the plant response consists of early and late responses as well as memory reprogramming for long-term protection against cold stress. The regulatory network can be differentiated into CBF-dependent and independent pathways involving different sets of TFs. Post-transcriptional regulation by miRNAs, control during ribosomal translation process, and post-translational regulation involving 26S proteosomic degradation are processes that affect the cellular abundance of key regulatory TFs, which is an important aspect of the regulation for cold acclimation. Therefore, fine-tuning of the regulation by TFs for adjusting to the cold stress condition involving the dynamic action of protein kinases, membrane ion channels, adapters, and modifiers is emphasized in this review.
Plants have the ability to show responses against various environmental stresses.
It is one of the necessities to understand stress response mechanisms to improve
crops productivity and quality, under the stressed condition.
The AP2/ERF transcription factors are one of the putative candidates that are
involved in the regulation of biotic and abiotic stress. Most of the research has
been conducted on functional analysis of AP2/ERF genes in many plants;
however, a comprehensive review is required to show a broad picture of
functionally characterized AP2/ERF in different plants. In this study, a
comprehensive review is carried on genome-wide studies of AP2/ERF gene
family and their evolutionary divergence in plant species including mustard
(Arabidopsis, brassica), cereal (rice, wheat, maize, sorghum), and fiber (upland
cotton and island cotton). Review exhibited that AP2/ERF superfamily is
classified into four sub-families e.g. AP2, DREB, ERF, RAV and solicit, in which the
ERF was the largest sub-family of AP2/ERF superfamily. Each subfamily was
further divided into multiple groups and sub-groups. Furthermore, each plant
species showed different number of paralogs showing correspondence to the
plant genome size .e.g. higher genome possess higher gene copy number. The
change in copy number may be due to either tandem gene duplication or whole
genome duplication during evolutionary adaptation that developed special
feature in plant species under environmental stresses. Moreover, current study
also surveyed on the expression of AP/ERF genes with the conclusion that
expression of AP2/ERF produced tolerance against biotic and abiotic stresses.
However further studies are required to improve crops resistance by studying
the same variables and gene families in different plants.
The apetala/ethylene responsive factor (AP2/ERF) family is one of the largest plant-specific transcription factors and plays a vital role in plant development and response to stress. The apetala 2.4 (RAP2.4) gene is a member of the AP2/ERF family. In this study, ClRAP2.4 cDNA fragment with 768bp open reading frame was cloned and the resistance of ClRAP2.4 overexpression to low temperature was investigated to understand whether RAP2.4 is involved in low-temperature stress in chrysanthemum (Chrysamthemum lavandulifolium). Phylogenetic analysis showed that ClRAP2.4 belonged to the DREB subfamily and was most closely related to AT1G22190. ClRAP2.4 was localised in cell nucleus and promotes transcriptional activation in yeast. In addition, ClRAP2.4 was transformed by using the Agrobacterium-mediated leaf disc method, and four overexpression lines (OX-1, OX-2, OX-7, and OX-8) were obtained. The activities of superoxide dismutase and peroxidase, and proline content in leaves in the four overexpression line were higher than those in the wild type (WT), whereas the electrical conductivity and malondialdehyde content were decreased, indicating that the tolerance of plants with ClRAP2.4 overexpression to cold stress was increased. RNA-Seq showed 390 differentially expressed genes (DEGs) between the transgenic and WT plants(229 upregulated, 161 downregulated). The number of ABRE, LTR, and DRE cis-elements in the promoters of DEGs were 175, 106, and 46, respectively. The relative expression levels of ClCOR, ClFe/MnSOD, ClPOD, ClNCL, ClPLK, ClFAD, and ClPRP in the transgenic plants were higher than those in WT plants at low temperatures. These data suggest that ClRAP2.4 may increase chrysanthemum tolerance to cold stress.
Osmotic stress frequently caused by drought is the most threatening environmental stress that remarkably reduces crop yield. Oat ( Avena sativa L.) is sensitive to water deficiency during growth. Plant metallothioneins (pMTs) show tremendous promise in enhancing general stress tolerance in plants. This study aimed to verify whether pMTs and elements of the antioxidant defence system protect oat against osmotic stress. Coding and genomic regions of A. sativa L. MTs belonging to three different types were cloned. To evaluate the role of MTs in osmotic stress, the expression of genes encoding AsMT1‐3 was checked by qRT‐PCR in the roots and shoots of oat plants growing in a hydroponic culture in the presence of polyethylene glycol (PEG 6000) and mannitol. The expression of AsMT1‐3 changed in response to osmotic stress; however, the changes depended on the type of MT and the treatment. The amount of AsMT3 transcript was about five‐fold and nine‐fold higher in shoots and roots, respectively, in the presence of mannitol. To further analyse the response of oat to osmotic stress, the level of phenolic compounds, soluble sugars, abscisic acid, and the activity of antioxidant enzymes were tested using spectrophotometric and chromatographic methods. During osmotic stress, the content of phenols, soluble sugars, abscisic acid, and the activity of catalase and peroxidase in shoots increased. In roots treated with PEG 6000, the amount of phenolic compounds was higher than that in roots treated with mannitol. The activity of superoxide dismutase was about 5‐fold higher in roots than in shoots. MTs are involved in plant response to osmotic stress. In the future, insight provided in this study will lead to application in agriculture either by using MTs as molecular markers for stress‐resistant crop cultivars or by a generation of genetically modified crops overexpressing MT s.
Cellobiose is the primary product of cellulose hydrolysis and is expected to be served as pathogen/damage-associated molecular patterns (PAMPs/DAMPs) in evoking plant innate immunity. In the study, cellobiose was demonstrated to be a positive regulator in immune response, but devote to halt autoimmunity when lettuce was exposed to cellobiose higher than 60 mg·L –1. When infected by Botrytis cinerea, cellobiose endowed plants with enhanced pre-invasion resistance by activating high levels of β-1,3-glucanase and anti-oxidative enzyme activities at the initial stage of pathogenic infection. Meanwhile, cellobiose-activated core regulatory factors such as EDS1, PTI6, and WRKY70 as well as SA signaling played an indispensable role in modulating plant growth-defense tradeoffs. Transcriptomics data further suggested that the cellobiose-activated plant-pathogen pathways are involved in MAMPs / PAMPs-triggered immune responses (PTI). Genes encoding receptor-like kinases, redox homeostasis, phytohormone signal transduction, transcription factors, and pathogenesis-related proteins were also detected to be up- or down-regulated by cellobiose. Taken together, this study demonstrated that cellobiose serves as an elicitor can directly activate disease resistance-related cellular functions. Besides, multiple genes have been identified as potential modulators of the cellobiose-induced immune response, which could inform efforts to understand underlying molecular events.
Objective
AP2/ERF (APETALA2/ethylene-responsive factor) superfamily is one of the largest gene families in plants and has been reported to participate in various biological processes, such as the regulation of biosynthesis of active lignan. However, few studies have investigated the genome-wide role of the AP2/ERF superfamily in Isatis indigotica. This study establishes a complete picture of the AP2/ERF superfamily in I. indigotica and contributes valuable information for further functional characterization of IiAP2/ERF genes and supports further metabolic engineering.
Methods
To identify the IiAP2/ERF superfamily genes, the AP2/ERF sequences from Arabidopsis thaliana and Brassica rapa were used as query sequences in the basic local alignment search tool. Bioinformatic analyses were conducted to investigate the protein structure, motif composition, chromosome location, phylogenetic relationship, and interaction network of the IiAP2/ERF superfamily genes. The accuracy of omics data was verified by quantitative polymerase chain reaction and heatmap analyses.
Results
One hundred and twenty-six putative IiAP2/ERF genes in total were identified from the I. indigotica genome database in this study. By sequence alignment and phylogenetic analysis, the IiAP2/ERF genes were classified into 5 groups including AP2, ERF, DREB (dehydration-responsive element-binding factor), Soloist and RAV (related to abscisic acid insensitive 3/viviparous 1) subfamilies. Among which, 122 members were unevenly distributed across seven chromosomes. Sequence alignment showed that I. indigotica and A. thaliana had 30 pairs of orthologous genes, and we constructed their interaction network. The comprehensive analysis of gene expression pattern in different tissues suggested that these genes may play a significant role in organ growth and development of I. indigotica. Members that may regulate lignan biosynthesis in roots were also preliminarily identified. Ribonucleic acid sequencing analysis revealed that the expression of 76 IiAP2/ERF genes were up- or down-regulated under salt or drought treatment, among which, 33 IiAP2/ERF genes were regulated by both stresses.
Conclusion
This study undertook a genome-wide characterization of the AP2/ERF superfamily in I. indigotica, providing valuable information for further functional characterization of IiAP2/ERF genes and discovery of genetic targets for metabolic engineering.
Climate change leads to various abiotic stresses that the plants face within their life cycles. This leads to extensive biochemical, physiological, and morphological changes in plants. Plants have evolved several defense mechanisms to survive in abiotic stress conditions, and all of these mechanisms include the differential expression of hundreds of genes involved in tens of different biological pathways. Recent systems biology approaches have identified a core gene network that is essential in abiotic stress tolerance in plants. However, a more detailed analysis is required to understand the core transcriptional regulatory network of stress-responsive genes in the model plant Arabidopsis thaliana. Here we explain the identification of a core transcriptional regulatory network of stress-responsive genes in Arabidopsis by bioinformatic analyses under several abiotic stress conditions. Then, known functions of identified transcription factor families are discussed in detail.KeywordsAbiotic stress
Arabidopsis thaliana
Climate changeGene networksGenetic engineeringTranscription factors
The apetala/ethylene responsive factor (AP2/ERF) family is one of the largest plant-specific transcription factors and plays a vital role in plant development and response to stress. The apetala 2.4 (RAP2.4) gene is a member of the AP2/ERF family. Whether RAP2.4 is involved in low-temperature stress in chrysanthemum is unknown. Here, we cloned the ClRAP2.4 cDNA fragment containing an open reading frame of 768 bp. Phylogenetic analysis showed that ClRAP2.4 was most closely related to AT1G22190 and belonged to the DREB subfamily. The ClRAP2.4 protein is localized in the cell nucleus and promotes transcriptional activation in yeast. We obtained four overexpression lines (OX-1, OX-2, OX-7, and OX-8) using the Agrobacterium-mediated leaf disc method. The overexpression of ClRAP2.4 in chrysanthemum increased tolerance to cold stress. The activities of superoxide dismutase (SOD) and peroxidase (POD) and the proline content in the transgenic chrysanthemum leaf were higher than those in the wild type (WT) under cold stress, but electrical conductivity and malondialdehyde (MDA) content in transgenic plants increased rapidly with decreasing temperature.We have identified 390 differentially expressed genes (DEGs) between the transgenic plants and wild type using RNA-Seq, among which, 229 were upregulated and 161 were downregulated. The number of ABRE , LTR , and DRE cis-elements in the DEGs promoter were 175, 106, and 46, respectively. The relative expression levels of ClCOR , ClAPX , ClAP , ClNCL , ClPLK , ClFAD , ClMYB in transgenic plants were higher than those in WT plants at low temperatures. Collectively, these data suggest that ClRAP2.4 may increase chrysanthemum tolerance to cold stress.
Mevcut çalışmanın temel amacı, tuz stresi altındaki mısır fidelerine aseton O-(4 klorofenilsülfonil) oksim (AO) molekülünün ön muamelesinin stresin olumsuz etkilerini hafifletici etkilerinin olup olmadığının araştırılmasıdır. Bunun için; 18 saat distile su kontrol (K), 6 saat AO+12 saat distile su (AO), 6 saat distile su+12 saat 100 mM NaCl (TS) ve 6 saat AO+12 saat 100 mM NaCl (AO+TS) deney düzeneği kurulmuştur. Elde edilen bulgulara göre; kontrol uygulaması ile AO uygulaması arasında nispi su içeriği (NSİ) açısından bir fark saptanamazken, TS’de ciddi bir düşüş AO+TS’de ise kontrole göre önemli bir artış olduğu belirlendi. Klorofil içeriği TS uygulamasında AO ve kontrole göre azalırken, AO+TS uygulamasında içerik TS’ye göre önemli bir artış gösterdi. En yüksek karotenoid içeriği TS uygulamasında görülürken, en düşük içerik AO+TS’de belirlendi. MDA ve H2O2 içeriklerinde AO uygulamasında kontrole göre önemli bir azalış gözlenirken, TS’de kontrole göre ciddi bir artış AO+TS’de ise TS ile kıyaslandığında önemli bir azalış belirlendi. Guaiacol peroksidaz, katalaz, askorbat peroksidaz ve süperoksit dismutaz enzimleri AO ön uygulaması ile aktivitelerini düzenleyerek MDA ve H2O2 içeriğini önemli ölçüde azalttığı belirlendi. AO uygulaması ile prolin içeriğinde kontrole göre önemli bir artış gözlenirken, AO+TS’nin TS uygulamasına göre içerikte önemli bir azalışa neden olduğu belirlendi. AO uygulamasının fenolik madde içeriği üzerinde önemli değişikliklere neden olduğu gözlendi. Elde edilen bulgular ışığında, tuz stresi altındaki mısır fidelerine AO ön uygulamasının metabolizmanın genel işleyişini engelleme potansiyeline sahip radikallerin oluşumunu engelleyebileceğini düşündürmektedir.
Transcription factors (TFs) are key nodes of gene regulatory networks that specify plant morphogenesis and control specific pathways such as stress responses. TFs directly interact the genome by recognizing specific DNA sequence, in terms of a complex system to fine-tune spatiotemporal gene expression. The combinatorial interaction among TFs determines regulatory specificity and defines the set of target genes to orchestrate their expression during developmental switches. In this chapter, we provide a catalog of plant-specific TFs and a comprehensive assessment of whether genome-wide analyses have so far been used for identifying potential direct target genes for each TFs. We further construct comprehensive TF-associated regulatory networks in the model plant Arabidopsis thaliana using genome-wide datasets from our ChIP-Hub database (http://www.chiphub.org/). We discuss how to dissect the network structure to identify potentially important cross-regulatory loops in the control of developmental switches in plants.
Inflictions caused by cold stress can result in disastrous effects on the productivity and survival of plants. Cold stress response in plants requires crosstalk between multiple signaling pathways including cold, heat, and reactive oxygen species (ROS) signaling networks. CBF, MYB, bHLH, and WRKY families are among the TFs that function as key players in the regulation of cold stress response at the molecular level. This review discusses some of the latest understanding on the regulation of expression and the mechanistic actions of plant TFs to address cold stress response. It was shown that the plant response consists of early and late responses as well as memory reprogramming for long-term protection against cold stress. The regulatory network can be differentiated into CBF-dependent and independent pathways involving different sets of TFs. Post-transcriptional regulation by miRNAs, control during ribosomal translation process, and post-translational regulation involving 26S proteosomic degradation are processes that affect the cellular abundance of key regulatory TFs, which is an important aspect of the regulation for cold acclimation. Therefore, fine-tuning of the regulation by TFs for adjusting to the cold stress condition involving the dynamic action of protein kinases, membrane ion channels, adapters, and modifiers is emphasized in this review.
AP2/ERF (APETALA2/ethylene-responsive factor) family transcription factors are involved in various plant-specific processes, especially in plant development and response to abiotic stress. However, their roles in thermotolerance are still largely unknown. In the current study, we identified a heat-inducible ERF member LlERF110 from Lilium longiflorum that was rapidly induced by high temperature. Its protein was localized in the nucleus, and transcriptional activation activity was observed in yeast and plant cells. In addition, LlERF110 was able to bind to GCC- and CGG-elements, but not to DRE-elements. Overexpression of LlERF110 conferred delayed bolting and bushy phenotype, with decreased thermotolerance accompanied by a disrupted ROS (reactive oxygen species) homeostasis in transgenic plants. The accumulation of LlERF110 may activate certain repressors related to heat stress response (HSR) and indirectly damage the normal expression of heat stress (HS)-protective genes such as AtHSFA2, which consequently leads to reduced thermotolerance. Our results implied that LlERF110 might function as a heat-inducible gene but may hinder the establishment of thermotolerance.
Salt stress causes the quality change and significant yield loss of tomato. However, the resources of salt-resistant tomato were still deficient and the mechanisms of tomato resistance to salt stress were still unclear. In this study, the proteomic profiles of two salt-tolerant and salt-sensitive tomato cultivars were investigated to decipher the salt-resistance mechanism of tomato and provide novel resources for tomato breeding. We found high abundance proteins related to nitrate and amino acids metabolismsin the salt-tolerant cultivars. The significant increase in abundance of proteins involved in Brassinolides and GABA biosynthesis were verified in salt-tolerant cultivars, strengthening the salt resistance of tomato. Meanwhile, salt-tolerant cultivars with higher abundance and activity of antioxidant-related proteins have more advantages in dealing with reactive oxygen species caused by salt stress. Moreover, the salt-tolerant cultivars had higher photosynthetic activity based on overexpression of proteins functioned in chloroplast, guaranteeing the sufficient nutrient for plant growth under salt stress. Furthermore, three key proteins were identified as important salt-resistant resources for breeding salt-tolerant cultivars, including sterol side chain reductase, gamma aminobutyrate transaminase and starch synthase. Our results provided series valuable strategies for salt-tolerant cultivars which can be used in future.
The monoterpene linalool is a major contributor to flavor of multiple fruit species. Although great progress has been made in identifying genes related to linalool formation, transcriptional regulation for the pathway remains largely unknown. As a super transcription factor family, roles of AP2/ERF in regulating linalool production have not been elucidated. Peach linalool is catalyzed by terpene synthase PpTPS1 and PpTPS3. Here, we observed that expression of PpERF61 correlated with these two PpTPSs during fruit ripening by transcriptome co-expression analysis. Dual-luciferase assay and EMSA results indicated that PpERF61 activated the PpTPS1 and PpTPS3 transcription by binding to the DRE/CRT motif in their promoters. Transient overexpressing PpERF61 in peach fruit significantly increased PpTPS1 and PpTPS3 expression and linalool content. Further study revealed significant correlation between PpERF61 transcripts and linalool contents across 30 peach cultivars. Besides transcriptional regulation, accumulated linalool was associated with DNA demethylation of PpERF61 during peach fruit ripening. In addition, interactions between PpERF61 and PpbHLH1 were evaluated, indicating these two transcription factors were associated with linalool production during peach fruit ripening. Overall, our results revealed a new insight into the regulation of linalool synthesis in fruit.
Key message
Transcriptomes of solanaceous plants expressing a plastid-targeted antioxidant protein were analysed to identify chloroplast redox networks modulating the expression of nuclear genes associated with stress acclimation.
Abstract
Plastid functions depend on the coordinated expression of nuclear genes, many of them associated to developmental and stress response pathways. Plastid-generated signals mediate this coordination via retrograde signaling, which includes sensing of chloroplast redox state and levels of reactive oxygen species (ROS), although it remains a poorly understood process. Chloroplast redox poise and ROS build-up can be modified by recombinant expression of a plastid-targeted antioxidant protein, i.e., cyanobacterial flavodoxin, with the resulting plants displaying increased tolerance to multiple environmental challenges. Here we analysed the transcriptomes of these flavodoxin-expressing plants to study the coordinated transcriptional responses of the nucleus to the chloroplast redox status and ROS levels during normal growth and stress responses (drought or biotic stress) in tobacco and potato, members of the economically important Solanaceae family. We compared their transcriptomes against those from stressed and mutant plants accumulating ROS in different subcellular compartments and found distinct ROS-related imprints modulated by flavodoxin expression and/or stress. By introducing our datasets in a large-scale interaction network, we identified transcriptional factors related to ROS and stress responses potentially involved in flavodoxin-associated signaling. Finally, we discovered identical cis elements in the promoters of many genes that respond to flavodoxin in the same direction as in wild-type plants under stress, suggesting a priming effect of flavodoxin before stress manifestation. The results provide a genome-wide picture illustrating the relevance of chloroplast redox status on biotic and abiotic stress responses and suggest new cis and trans targets to generate stress-tolerant solanaceous crops.
The mode of abscisic acid (ABA) action, and its relations to drought adaptive responses in particular, has been a captivating area of plant hormone research for much over a decade. The hormone triggers stomatal closure to limit water loss through transpiration, as well as mobilizes a battery of genes that presumably serve to protect the cells from the ensuing oxidative damage in prolonged stress. The signaling network orchestrating these various responses is, however, highly complex. This review summarizes several significant advances made within the last few years. The biosynthetic pathway of the hormone is now almost completely elucidated, with the latest identification of the ABA4 gene encoding a neoxanthin synthase, which seems essential for de novo ABA biosynthesis during water stress. This leads to the interesting question on how ABA is then delivered to perception sites. In this respect, regulated transport has attracted renewed focus by the unexpected finding of a shoot-to-root translocation of ABA during drought response, and at the cellular level, by the identification of a beta-galactosidase that releases biologically active ABA from inactive ABA-glucose ester. Surprising candidate ABA receptors were also identified in the form of the Flowering Time Control Protein A (FCA) and the Chloroplastic Magnesium Protoporphyrin-IX Chelatase H subunit (CHLH) in chloroplast-nucleus communication, both of which have been shown to bind ABA in vitro. On the other hand, the protein(s) corresponding to the physiologically detectable cell-surface ABA receptor(s) is (are) still not known with certainty. Genetic and physiological studies based on the guard cell have reinforced the central importance of reversible phosphorylation in modulating rapid ABA responses. Sucrose Non-Fermenting Related Kinases (SnRK), Calcium-Dependent Protein Kinases (CDPK), Protein Phosphatases (PP) of the 2C and 2A classes figure as prominent regulators in this single-cell model. Identifying their direct in vivo targets of regulation, which may include H(+)-ATPases, ion channels, 14-3-3 proteins and transcription factors, will logically be the next major challenge. Emerging evidence also implicates ABA as a repressor of innate immune response, as hinted by the highly similar roster of genes elicited by certain pathogens and ABA. Undoubtedly, the most astonishing revelation is that ABA is not restricted to plants and mosses, but overwhelming evidence now indicates that it also exists in metazoans ranging from the most primitive to the most advance on the evolution scale (sponges to humans). In metazoans, ABA has healing properties, and plays protective roles against both environmental and pathogen related injuries. These cross-kingdom comparisons have shed light on the surprising ancient origin of ABA and its attendant mechanisms of signal transduction.
Abiotic stresses, such as drought and salinity, lead to crop growth damage and a decrease in crop yields. Stomata control CO(2) uptake and optimize water use efficiency, thereby playing crucial roles in abiotic stress tolerance. Hydrogen peroxide (H(2)O(2)) is an important signal molecule that induces stomatal closure. However, the molecular pathway that regulates the H(2)O(2) level in guard cells remains largely unknown. Here, we clone and characterize DST (drought and salt tolerance)-a previously unknown zinc finger transcription factor that negatively regulates stomatal closure by direct modulation of genes related to H(2)O(2) homeostasis-and identify a novel pathway for the signal transduction of DST-mediated H(2)O(2)-induced stomatal closure. Loss of DST function increases stomatal closure and reduces stomatal density, consequently resulting in enhanced drought and salt tolerance in rice. These findings provide an interesting insight into the mechanism of stomata-regulated abiotic stress tolerance, and an important genetic engineering approach for improving abiotic stress tolerance in crops.
ATTED-II (http://atted.jp) is a database of gene coexpression in Arabidopsis that can be used to design a wide variety of experiments, including the
prioritization of genes for functional identification or for studies of regulatory relationships. Here, we report updates
of ATTED-II that focus especially on functionalities for constructing gene networks with regard to the following points: (i)
introducing a new measure of gene coexpression to retrieve functionally related genes more accurately, (ii) implementing clickable
maps for all gene networks for step-by-step navigation, (iii) applying Google Maps API to create a single map for a large
network, (iv) including information about protein–protein interactions, (v) identifying conserved patterns of coexpression
and (vi) showing and connecting KEGG pathway information to identify functional modules. With these enhanced functions for
gene network representation, ATTED-II can help researchers to clarify the functional and regulatory networks of genes in Arabidopsis.
Arabidopsis abscisic acid (ABA)-insensitive abi4 mutants have pleiotropic defects in seed development, including decreased sensitivity to ABA inhibition of germination and altered seed-specific gene expression. This phenotype is consistent with a role for ABI4 in regulating seed responses to ABA and/or seed-specific signals. We isolated the ABI4 gene by positional cloning and confirmed its identity by complementation analysis. The predicted protein product shows homology to a plant-specific family of transcriptional regulators characterized by a conserved DNA binding domain, the APETALA 2 domain. The single mutant allele identified has a single base pair deletion, resulting in a frameshift that should disrupt the C-terminal half of the protein but leave the presumed DNA binding domain intact. Expression analyses showed that despite the seed-specific nature of the mutant phenotype, ABI4 expression is not seed specific.
Ethylene-responsive element-binding proteins (EREBPs)have novel DNA-binding domains (ERF domains), which are widely conserved
in plants, and interact specifically with sequences containing AGCCGCC motifs (GCC box). Deletion experiments show that some
flanking region at the N terminus of the conserved 59-amino acid ERF domain is required for stable binding to the GCC box.
Three ERF domain-containing fragments of EREBP2, EREBP4, and AtERF1 from tobacco and Arabidopsis, bind to the sequence containing the GCC box with a high binding affinity in the pm range. The high affinity binding is conferred by a monomeric ERF domain fragment, and DNA truncation experiments show that
only 11-base pair DNA containing the GCC box is sufficient for stable ERF domain interaction. Systematic DNA mutation analyses
demonstrate that the specific amino acid contacts are confined within the 6-base pair GCCGCC region of the GCC box, and the
first G, the fourth G, and the sixth C exhibit highest binding specificity common in all three ERF domain-containing fragments
studied. Other bases within the GCC box exhibit modulated binding specificity varying from protein to protein, implying that
these positions are important for differential binding by different EREBPs. The conserved N-terminal half is likely responsible
for formation of a stable complex with the GCC box and the divergent C-terminal half for modulating the specificity.
Transcription factors containing a conserved DNA-binding domain similar to that of the proto-oncogene c-myb have been identified in nearly all eukaryotes. MYB-related proteins from plants generally contain two related helix-turn-helix motifs, the R2 and R3 repeats. It was estimated that Arabidopsis thaliana contains more than 100 R2R3-MYB genes. The few cases where functional data are available suggest an important role of these genes in the regulation of secondary metabolism, the control of cell shape, disease resistance, and hormone responses. To determine the full regulatory potential of this large family of regulatory genes, a systematic search for the function of all genes of this family was initiated. Sequence data for more than 90 different A. thaliana R2R3-MYB genes have been obtained. Sequence comparison revealed conserved amino acid motifs shared by subgroups of R2R3-MYB genes in addition to the characteristic DNA-binding domain. No significant clustering of the genes was detected, although they are not uniformly distributed throughout the A. thaliana genome.
The Arabidopsis abscisic acid (ABA)-insensitive abi5 mutants have pleiotropic defects in ABA response, including decreased sensitivity to ABA inhibition of germination and altered expression of some ABA-regulated genes. We isolated the ABI5 gene by using a positional cloning approach and found that it encodes a member of the basic leucine zipper transcription factor family. The previously characterized abi5-1 allele encodes a protein that lacks the DNA binding and dimerization domains required for ABI5 function. Analyses of ABI5 expression provide evidence for ABA regulation, cross-regulation by other ABI genes, and possibly autoregulation. Comparison of seed and ABA-inducible vegetative gene expression in wild-type and abi5-1 plants indicates that ABI5 regulates a subset of late embryogenesis-abundant genes during both developmental stages.
We have characterized developmental, environmental, and genetic regulation of abscisic acid-insensitive (ABI)4 gene expression in Arabidopsis. Although expressed most strongly in seeds, ABI4 transcripts are also present at low levels in vegetative tissue; vegetative expression is not induced by abscisic acid (ABA) or stress treatments. Comparison of transcript levels in mature seeds of ABA-insensitive, ABA-hypersensitive, ABA-deficient, or heterochronic mutants indicates that ABI4 expression is altered in only two of the backgrounds, the ABA-insensitive mutants abi1-1 and abi3-1. To determine whether ABI4 is necessary and/or sufficient for ABA response, we assayed the effects of loss of ABI4 function and ectopic ABI4 expression on growth and gene expression. We examined genetic interactions among three ABA response loci, ABI3, ABI4, and ABI5, by comparing phenotypes of mutants, ectopic expression lines, mutants carrying an ectopically expressed transgene, and the corresponding wild-type lines. Our results indicate some cross-regulation of expression among ABI3, ABI4, and ABI5 and suggest that they function in a combinatorial network, rather than a regulatory hierarchy, controlling seed development and ABA response.
The completion of the Arabidopsis thaliana genome sequence allows a comparative analysis of transcriptional regulators across the three eukaryotic kingdoms. Arabidopsis dedicates over 5% of its genome to code for more than 1500 transcription factors, about 45% of which are from families specific
to plants.Arabidopsis transcription factors that belong to families common to all eukaryotes do not share significant similarity with those of
the other kingdoms beyond the conserved DNA binding domains, many of which have been arranged in combinations specific to
each lineage. The genome-wide comparison reveals the evolutionary generation of diversity in the regulation of transcription.
Using mRNA differential display analysis, we isolated a salt-induced transcript that showed a significant sequence homology with an EREBP/AP2 DNA binding motif from oilseed rape plants. With this cDNA fragment as a probe, cDNA clone Tsi1 (for Tobacco stress-induced gene1) was isolated from a tobacco cDNA library. RNA gel blot analysis indicated that transcripts homologous with Tsi1 were induced not only in NaCl-treated leaves but also in leaves treated with ethephon or salicylic acid. Transient expression analysis using a Tsi1::smGFP fusion gene in BY-2 cells indicated that the Tsi1 protein was targeted to the nucleus. Fusion protein of Tsi1 with GAL4 DNA binding domain strongly activated transcription in yeast, and the transactivating activity was localized to the 13 C-terminal amino acids of Tsi1. Electrophoretic mobility shift assays revealed that Tsi1 could bind specifically to the GCC and the DRE/CRT sequences, although the binding activity to the former was stronger than that to the latter. Furthermore, Agrobacterium-mediated transient expression and transgenic plants expressing Tsi1 demonstrated that overexpression of the Tsi1 gene induced expression of several pathogenesis-related genes under normal conditions, resulting in improved tolerance to salt and pathogens. These results suggest that Tsi1 might be involved as a positive trans-acting factor in two separate signal transduction pathways under abiotic and biotic stress.
Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.
Mechanical wounding not only damages plant tissues, but also provides pathways for pathogen invasion. To understand plant responses to wounding at a genomic level, we have surveyed the transcriptional response of 8,200 genes in Arabidopsis plants. Approximately 8% of these genes were altered by wounding at steady-state mRNA levels. Studies of expression patterns of these genes provide new information on the interactions between wounding and other signals, including pathogen attack, abiotic stress factors, and plant hormones. For example, a number of wound-responsive genes encode proteins involved in pathogen response. These include signaling molecules for the pathogen resistance pathway and enzymes required for cell wall modification and secondary metabolism. Many osmotic stress- and heat shock-regulated genes were highly responsive to wounding. Although a number of genes involved in ethylene, jasmonic acid, and abscisic acid pathways were activated, many in auxin responses were suppressed by wounding. These results further dissected the nature of mechanical wounding as a stress signal and identified new genes that may play a role in wounding and other signal transduction pathways.
Significant progress has been made in elucidating the mechanism of abscisic acid (ABA)-regulated gene expression, including the characterization of an ABA-responsive element (ABRE), which is regulated by basic domain/Leu zipper transcription factors. In addition to the ABRE, a coupling element (CE1) has been demonstrated to be involved in ABA-induced expression. However, a trans factor that interacts with CE1 has yet to be characterized. We report the isolation of a seed-specific maize ABI4 homolog and demonstrate, using a PCR-based in vitro selection procedure, that the maize ABI4 protein binds to the CE-1 like sequence CACCG. Using electrophoretic mobility shift assays, we demonstrate that recombinant ZmABI4 protein binds to the CE1 element in a number of ABA-related genes. ZmABI4 also binds to the promoter of the sugar-responsive ADH1 gene, demonstrating the ability of this protein to regulate both ABA- and sugar-regulated pathways. ZmABI4 complements Arabidopsis ABI4 function, because abi4 mutant plants transformed with the ZmABI4 gene have an ABA- and sugar-sensitive phenotype. Identification of the maize ABI4 ortholog and the demonstration of its binding to a known ABA response element provide a link between ABA-mediated kernel development and the regulation of ABA response genes.
Plants respond to environmental stress by activating "stress genes." The plant hormone abscisic acid (ABA) plays an important role in stress-responsive gene expression. Although Ca(2+) serves as a common second messenger in signaling stress and ABA, little is known about the molecular basis of Ca(2+) action in these pathways. Here, we show that CIPK3, a Ser/Thr protein kinase that associates with a calcineurin B-like calcium sensor, regulates ABA response during seed germination and ABA- and stress-induced gene expression in Arabidopsis. The expression of the CIPK3 gene itself is responsive to ABA and stress conditions, including cold, high salt, wounding, and drought. Disruption of CIPK3 altered the expression pattern of a number of stress gene markers in response to ABA, cold, and high salt. However, drought-induced gene expression was not altered in the cipk3 mutant plants, suggesting that CIPK3 regulates select pathways in response to abiotic stress and ABA. These results identify CIPK3 as a molecular link between stress- and ABA-induced calcium signal and gene expression in plant cells. Because the cold signaling pathway is largely independent of endogenous ABA production, CIPK3 represents a cross-talk "node" between the ABA-dependent and ABA-independent pathways in stress responses.
To elucidate gene function on a global scale, we identified pairs of genes that are coexpressed over 3182 DNA microarrays
from humans, flies, worms, and yeast. We found 22,163 such coexpression relationships, each of which has been conserved across
evolution. This conservation implies that the coexpression of these gene pairs confers a selective advantage and therefore
that these genes are functionally related. Manyof these relationships provide strong evidence for the involvement of new genes
in core biological functions such as the cell cycle, secretion, and protein expression. We experimentallyconfirmed the predictions
implied bysome of these links and identified cell proliferation functions for several genes. By assembling these links into
a gene-coexpression network, we found several components that were animal-specific as well as interrelationships between newly
evolved and ancient modules.
Plant growth and development are regulated by internal signals and by external environmental con- ditions. One important regulator that coordinates growth and development with responses to the en- vironment is the sesquiterpenoid hormone abscisic acid (ABA). ABA plays important roles in many cel- lular processes including seed development, dor- mancy, germination, vegetative growth, and environ- mental stress responses. These diverse functions of ABA involve complex regulatory mechanisms that control its production, degradation, signal percep- tion, and transduction. Because of the key role of ABA in plant stress responses, understanding these regulatory mechanisms will help devise rational strategies to breed or genetically engineer crop plants with increased tolerance to adverse environmental conditions. Since the discovery of ABA in the early 1960s, much effort has been devoted to understanding how ABA is synthesized. Through genetic and biochemi- cal studies, the pathway for ABA biosynthesis in higher plants is now understood in great detail. Re- cently, all the major genes for the enzymes in the biosynthesis pathway have been identified (Schwartz et al., 2003). The new challenge is to understand how these biosynthesis genes, and the biosynthetic path- way as a whole, are regulated. Although much re- mains to be learned about the regulatory mechanism, evidence thus far indicates that ABA biosynthesis is subject to complex regulation during plant develop- ment and in response to environmental stresses. In this Update, we first present a brief overview of the functions of ABA and the biosynthesis pathway. We then focus on the regulation of ABA production and attempt to provide some future directions in ABA biosynthesis studies.
Abscisic acid (ABA) regulates many aspects of plant growth and development, yet many ABA response mutants present only subtle phenotypic defects, especially in the absence of stress. By contrast, the ABA-insensitive8 (abi8) mutant, isolated on the basis of ABA-resistant germination, also displays severely stunted growth, defective stomatal regulation, altered ABA-responsive gene expression, delayed flowering, and male sterility. The stunted growth of the mutant is not rescued by gibberellin, brassinosteroid, or indoleacetic acid application and is not attributable to excessive ethylene response, but supplementing the medium with Glc improves viability and root growth. In addition to exhibiting Glc-dependent growth, reflecting decreased expression of sugar-mobilizing enzymes, abi8 mutants are resistant to Glc levels that induce developmental arrest of wild-type seedlings. Studies of genetic interactions demonstrate that ABA hypersensitivity conferred by the ABA-hypersensitive1 mutation or overexpression of ABI3 or ABI5 does not suppress the dwarfing and Glc dependence caused by abi8 but partially suppresses ABA-resistant germination. By contrast, the ABA-resistant germination of abi8 is epistatic to the hypersensitivity caused by ethylene-insensitive2 (ein2) and ein3 mutations, yet ABI8 appears to act in a distinct Glc response pathway from these EIN loci. ABI8 encodes a protein with no domains of known function but belongs to a small plant-specific protein family. Database searches indicate that it is allelic to two dwarf mutants, elongation defective1 and kobito1, previously shown to disrupt cell elongation, cellulose synthesis, vascular differentiation, and root meristem maintenance. The cell wall defects appear to be a secondary effect of the mutations because Glc treatment restores root growth and vascular differentiation but not cell elongation. Although the ABI8 transcript accumulates in all tested plant organs in both wild-type and ABA response mutants, an ABI8-beta-glucuronidase fusion protein is localized primarily to the elongation zone of roots, suggesting substantial post-transcriptional regulation of ABI8 accumulation. This localization pattern is sufficient to complement the mutation, indicating that ABI8 acts either at very low concentrations or over long distances within the plant body.
The plant hormone abscisic acid (ABA) plays a major role in seed maturation and germination, as well as in adaptation to abiotic environmental stresses. ABA promotes stomatal closure by rapidly altering ion fluxes in guard cells. Other ABA actions involve modifications of gene expression, and the analysis of ABA-responsive promoters has revealed a diversity of potential cis-acting regulatory elements. The nature of the ABA receptor(s) remains unknown. In contrast, combined biophysical, genetic, and molecular approaches have led to considerable progress in the characterization of more downstream signaling elements. In particular, substantial evidence points to the importance of reversible protein phosphorylation and modifications of cytosolic calcium levels and pH as intermediates in ABA signal transduction. Exciting advances are being made in reassembling individual components into minimal ABA signaling cascades at the single-cell level.
The phytohormone abscisic acid (ABA) plays an essential role in adaptive stress responses. The hormone regulates, among others, the expression of numerous stress-responsive genes. From various promoter analyses, ABA-responsive elements (ABREs) have been determined and a number of ABRE binding factors have been isolated, although their in vivo roles are not known. Here we report that the ABRE binding factors ABF3 and ABF4 function in ABA signaling. The constitutive overexpression of ABF3 or ABF4 in Arabidopsis resulted in ABA hypersensitivity and other ABA-associated phenotypes. In addition, the transgenic plants exhibited reduced transpiration and enhanced drought tolerance. At the molecular level, altered expression of ABA/stress-regulated genes was observed. Furthermore, the temporal and spatial expression patterns of ABF3 and ABF4 were consistent with their suggested roles. Thus, our results provide strong in vivo evidence that ABF3 and ABF4 mediate stress-responsive ABA signaling.
We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif L/FDLNL/F(x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.
Using mRNA differential display analysis, we isolated a salt-induced transcript that showed a significant sequence homology with an EREBP/AP2 DNA binding motif from oilseed rape plants. With this cDNA fragment as a probe, cDNA clone Tsi1 (for Tobacco stress-induced gene1) was isolated from a tobacco cDNA library. RNA gel blot analysis indicated that transcripts homologous with Tsi1 were induced not only in NaCl-treated leaves but also in leaves treated with ethephon or salicylic acid. Transient expression analysis using a Tsi1::smGFP fusion gene in BY-2 cells indicated that the Tsi1 protein was targeted to the nucleus. Fusion protein of Tsi1 with GAL4 DNA binding domain strongly activated transcription in yeast, and the transactivating activity was localized to the 13 C-terminal amino acids of Tsi1. Electrophoretic mobility shift assays revealed that Tsi1 could bind specifically to the GCC and the DRE/CRT sequences, although the binding activity to the former was stronger than that to the latter. Furthermore, Agrobacterium-mediated transient expression and transgenic plants expressing Tsi1 demonstrated that overexpression of the Tsi1 gene induced expression of several pathogenesis-related genes under normal conditions, resulting in improved tolerance to salt and pathogens. These results suggest that Tsi1 might be involved as a positive trans-acting factor in two separate signal transduction pathways under abiotic and biotic stress.
Ethylene-responsive element binding factors (ERFs) are members of a novel family of transcription factors that are specific to plants. A highly conserved DNA binding domain known as the ERF domain is the unique feature of this protein family. To characterize in detail this family of transcription factors, we isolated Arabidopsis cDNAs encoding five different ERF proteins (AtERF1 to AtERF5) and analyzed their structure, DNA binding preference, transactivation ability, and mRNA expression profiles. The isolated AtERFs were placed into three classes based on amino acid identity within the ERF domain, although all five displayed GCC box–specific binding activity. AtERF1, AtERF2, and AtERF5 functioned as activators of GCC box–dependent transcription in Arabidopsis leaves. By contrast, AtERF3 and AtERF4 acted as repressors that downregulated not only basal transcription levels of a reporter gene but also the transactivation activity of other transcription factors. The AtERF genes were differentially regulated by ethylene and by abiotic stress conditions, such as wounding, cold, high salinity, or drought, via ETHYLENE-INSENSITIVE2 (EIN2)–dependent or –independent pathways. Cycloheximide, a protein synthesis inhibitor, also induced marked accumulation of AtERF mRNAs. Thus, we conclude that AtERFs are factors that respond to extracellular signals to modulate GCC box–mediated gene expression positively or negatively.
We have characterized developmental, environmental, and genetic regulation of abscisic acid-insensitive (ABI)4 gene expression in Arabidopsis. Although expressed most strongly in seeds,ABI4 transcripts are also present at low levels in vegetative tissue; vegetative expression is not induced by abscisic acid (ABA) or stress treatments. Comparison of transcript levels in mature seeds of ABA-insensitive, ABA-hypersensitive, ABA-deficient, or heterochronic mutants indicates that ABI4 expression is altered in only two of the backgrounds, the ABA-insensitive mutantsabi1-1 and abi3-1. To determine whetherABI4 is necessary and/or sufficient for ABA response, we assayed the effects of loss of ABI4 function and ectopicABI4 expression on growth and gene expression. We examined genetic interactions among three ABA response loci,ABI3, ABI4, and ABI5, by comparing phenotypes of mutants, ectopic expression lines, mutants carrying an ectopically expressed transgene, and the corresponding wild-type lines. Our results indicate some cross-regulation of expression among ABI3, ABI4, andABI5 and suggest that they function in a combinatorial network, rather than a regulatory hierarchy, controlling seed development and ABA response.
The AP2 transcription factor family, found only in plants, includes several genes that encode proteins involved in the regulation of disease resistance pathways. These genes are members of the ethylene response factor (ERF) subfamily of AP2 transcription factor genes, which have only a single DNA-binding domain and are distinct from members of the dehydration-responsive element binding (DREB) subfamily. Some ERF subgroups are enriched in such genes, suggesting that they have conserved functions that are required for the regulation of disease resistance pathways. The expression of several ERF genes is regulated by plant hormones, such as jasmonic acid, salicylic acid and ethylene, as well as by pathogen challenge. A phylogenetic overview of these genes, with a focus on Arabidopsis, rice and tomato, suggests that despite broad conservation of their function in monocots and dicots, some structural elements are specialized within each of these two lineages.
Summary Transcription factors containing a conserved DNA-binding domain similar to that of the proto-oncogenec-mybhave been identified in nearly all eukaryotes. MYB-related proteins from plants generally contain two related helix-turn-helix motifs, the R2 and R3 repeats. It was estimated thatArabidopsis thalianacontains more than 100R2R3-MYBgenes. The few cases where functional data are available suggest an important role of these genes in the regulation of secondary metabolism, the control of cell shape, disease resistance, and hormone responses. To determine the full regulatory potential of this large family of regulatory genes, a systematic search for the function of all genes of this family was initiated.Sequence data for more than 90 differentA. thaliana R2R3-MYBgenes have been obtained. Sequence comparison revealed conserved amino acid motifs shared by subgroups ofR2R3-MYBgenes in addition to the characteristic DNA-binding domain. No significant clustering of the genes was detected, although they are not uniformly distributed throughout theA. thalianagenome.R2R3-MYBgene expression levels were determined under more than 20 different growth conditions including hormone treatment, infection with pathogens and various stress conditions.MYBgenes are specifically expressed in different tissues and physiological conditions, indicating the potential for involvement in various regulatory processes. The sequence and expression data together with the map positions of nearly allMYBgenes inA. thalianaprovide a substantial basis for further studies of this important group of transcription factors.
TheAgrobacteriumvacuum infiltration method has made it possible to transformArabidopsis thalianawithout plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containingAgrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, andAgrobacteriumcould be applied to plants at a range of cell densities. Repeated application ofAgrobacteriumimproved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation ofArabidopsisfor efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
The transcription factor ABA INSENSITIVE 4 (ABI4), discovered nearly 10 years ago, plays a central role in a variety of functions in plants, including sugar responses. However, not until very recently has its mechanism of action begun to be elucidated. Modulating gene expression is one of the primary mechanisms of sugar regulation in plants. Nevertheless, the transcription factors involved in regulating sugar responses and their role(s) during the signal transduction cascade remain poorly defined. In this paper we analyzed the participation of ABI4, as it is one of the main transcription factors implicated in glucose signaling during early seedling development. Our studies show that ABI4 is an essential activator of its own expression during development, in ABA signaling and in sugar responses. It is also important for the glucose-mediated expression of the genes ABI5 and SBE2.2. We demonstrate that ABI4 binds directly to the promoter region of all three genes and activates their expression in vivo through at CE1-like element. Previous studies found that ABI4 also functions as a transcriptional repressor of sugar-regulated genes, therefore this transcription factor is a versatile protein with dual functions for modulating gene expression.
A versatile gene expression cartridge and binary vector system was constructed for use in Agrobacterium-mediated plant transformation. The expression cartridge of the primary cloning vector, pART7, comprises of cauliflower mosaic virus Cabb B-JI isolate 35S promoter, a multiple cloning site and the transcriptional termination region of the octopine synthase gene. The entire cartridge can be removed from pART7 as a Not I fragment and introduced directly into the binary vector, pART27, recombinants being selected by blue/white screening for beta-galactosidase. pART27 carries the RK2 minimal replicon for maintenance in Agrobacterium, the ColE1 origin of replication for high-copy maintenance in Escherichia coli and the Tn7 spectinomycin/streptomycin resistance gene as a bacterial selectable marker. The organisational structure of the T-DNA of pART27 has been constructed taking into account the right to left border, 5' to 3' model of T-DNA transfer. The T-DNA carries the chimaeric kanamycin resistance gene (nopaline synthase promoter-neomycin phosphotransferase-nopaline synthase terminator) distal to the right border relative to the lacZ' region. Utilisation of these vectors in Agrobacterium-mediated transformation of tobacco demonstrated efficient T-DNA transfer to the plant genome.
We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
Ocs elements are a group of promoter sequences required for the expression of both pathogen genes in infected plants and plant defense genes. Genes for ocs element binding factors (OBFs), belonging to a specific class of basic-region leucine zipper (bZIP) transcription factors, have been isolated in a number of plants. Using protein-protein interaction screening with OBF4 we have isolated AtEBP, an Arabidopsis protein that contains a novel DNA-binding domain, the AP2/EREBP domain. One class of proteins that contain this domain are the tobacco ethylene-responsive element binding proteins (EREBPs). The EREBPs bind the GCC box that confers ethylene responsiveness to a number of pathogenesis related (PR) gene promoters. AtEBP expression is inducible by exogenous ethylene in wild-type plants and AtEBP transcripts are increased in the ctr1-1 mutant, where ethylene-regulated pathways are constitutively active. Electrophoretic mobility-shift assay and DNase I footprint analysis revealed that AtEBP can specifically bind to the GCC box. Interestingly, the highest level of AtEBP expression was detected in callus tissue, where ocs elements are very active. Synergistic effects of the GCC box with ocs elements or the related G-box sequence have been previously observed, for example, in the ethylene-induced expression of a PR gene promoter. Our results suggest that cross-coupling between EREBP and bZIP transcription factors occurs and may therefore be important in regulating gene expression during the plant defense response.
The rab17 gene from maize is transcribed in late embryonic development and is responsive to abscisic acid and water stress in embryo and vegetative tissues. In vivo footprinting and transient transformation of rab17 were performed in embryos and vegetative tissues to characterize the cis-elements involved in regulation of the gene. By in vivo footprinting, protein binding was observed to nine elements in the promoter, which correspond to five putative ABREs (abscisic acid responsive elements) and four other sequences. The footprints indicated that distinct proteins interact with these elements in the two developmental stages. In transient transformation, six of the elements were important for high level expression of the rab17 promoter in embryos, whereas only three elements were important in leaves. The cis-acting sequences can be divided in embryo-specific, ABA-specific and leaf-specific elements on the basis of protein binding and the ability to confer expression of rab17. We found one positive, new element, called GRA, with the sequence CACTGGCCGCCC. This element was important for transcription in leaves but not in embryos. Two other non-ABRE elements that stimulated transcription from the rab17 promoter resemble previously described abscisic acid and drought-inducible elements. There were differences in protein binding and function of the five ABREs in the rab17 promoter. The possible reasons for these differences are discussed. The in vivo data obtained suggest that an embryo-specific pathway regulates transcription of the rab genes during development, whereas another pathway is responsible for induction in response to ABA and drought in vegetative tissues.
A system of cluster analysis for genome-wide expression data from DNA microarray hybridization is described that uses standard statistical algorithms to arrange genes according to similarity in pattern of gene expression. The output is displayed graphically, conveying the clustering and the underlying expression data simultaneously in a form intuitive for biologists. We have found in the budding yeast Saccharomyces cerevisiae that clustering gene expression data groups together efficiently genes of known similar function, and we find a similar tendency in human data. Thus patterns seen in genome-wide expression experiments can be interpreted as indications of the status of cellular processes. Also, coexpression of genes of known function with poorly characterized or novel genes may provide a simple means of gaining leads to the functions of many genes for which information is not available currently.
The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
Ethylene-responsive element binding factors (ERFs) are members of a novel family of transcription factors that are specific to plants. A highly conserved DNA binding domain known as the ERF domain is the unique feature of this protein family. To characterize in detail this family of transcription factors, we isolated Arabidopsis cDNAs encoding five different ERF proteins (AtERF1 to AtERF5) and analyzed their structure, DNA binding preference, transactivation ability, and mRNA expression profiles. The isolated AtERFs were placed into three classes based on amino acid identity within the ERF domain, although all five displayed GCC box-specific binding activity. AtERF1, AtERF2, and AtERF5 functioned as activators of GCC box-dependent transcription in Arabidopsis leaves. By contrast, AtERF3 and AtERF4 acted as repressors that downregulated not only basal transcription levels of a reporter gene but also the transactivation activity of other transcription factors. The AtERF genes were differentially regulated by ethylene and by abiotic stress conditions, such as wounding, cold, high salinity, or drought, via ETHYLENE-INSENSITIVE2 (EIN2)-dependent or -independent pathways. Cycloheximide, a protein synthesis inhibitor, also induced marked accumulation of AtERF mRNAs. Thus, we conclude that AtERFs are factors that respond to extracellular signals to modulate GCC box-mediated gene expression positively or negatively.
Sugars have signaling roles in a wide variety of developmental processes in plants. To elucidate the regulatory components that constitute the glucose signaling network governing plant growth and development, we have isolated and characterized two Arabidopsis glucose insensitive mutants, gin5 and gin6, based on a glucose-induced developmental arrest during early seedling morphogenesis. The T-DNA-tagged gin6 mutant abrogates the glucose-induced expression of a putative transcription factor, ABI4, previously shown to be involved in seed-specific abscisic acid (ABA) responses. Thus, ABI4 might be a regulator involved in both glucose- and seed-specific ABA signaling. The characterization of the gin5 mutant, on the other hand, reveals that glucose-specific accumulation of ABA is essential for hexokinase-mediated glucose responses. Consistent with this result, we show that three ABA-deficient mutants (aba1-1, aba2-1, and aba3-2) are also glucose insensitive. Exogenous ABA can restore normal glucose responses in gin5 and aba mutants but not in gin6 plants. Surprisingly, only abi4 and abi5-1 but not other ABA-insensitive signaling mutants (abi1-1, abi2-1, and abi3-1) exhibit glucose insensitivity, indicating the involvement of a distinct ABA signaling pathway in glucose responses. These results provide the first direct evidence to support a novel and central role of ABA in plant glucose responses mediated through glucose regulation of both ABA levels by GIN5 and ABA signaling by GIN6/ABI4.
Crop plants are exposed to many types of abiotic stress during their life cycle. Water deficit derived from drought, low temperature or high salt concentration in the soil, is one of the most common environmental stresses that affects growth and development of plants through alterations in metabolism and gene expression. Adaptation to these conditions may involve passive tolerance or active homeostatic mechanisms for maintaining water balance. Active responses occur at different levels in the plant and may represent a concomitant protection against other types of stress such as pathogen attack. Many morphological and physiological adaptations to water stress are under the control of the plant hormone abscisic acid and involve specific activation of target genes that in one way or another protect cells against water deficit or participate in the regulation of the drought response. Here, we discuss recent advances in our understanding of drought adaptation mediated by specific changes in gene expression and the role of AP2/EREBP nuclear factors in these processes.
We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif (L)/(F)DLN(L)/(F)(x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.
DRE/CRT is a cis-acting element that is involved in gene expression responsive to drought and low-temperature stress in higher plants. DREB1A/CBF3 and DREB2A are transcription factors that specifically bind to DRE/CRT in Arabidopsis. We precisely analyzed the DNA-binding specificity of DREBs. Both DREBs specifically bound to six nucleotides (A/GCCGAC) of DRE. However, these proteins had different binding specificities to the second or third nucleotides of DRE. Gel mobility shift assay using mutant DREB proteins showed that the two amino acids, valine and glutamic acid conserved in the ERF/AP2 domains, especially valine, have important roles in DNA-binding specificity. In the Arabidopsis genome, 145 DREB/ERF-related proteins are encoded. These proteins were classified into five groups-AP-2 subfamily, RAV subfamily, DREB subfamily, ERF subfamily, and others. The DREB subfamily included three novel DREB1A- and six DREB2A-related proteins. We analyzed expression of novel genes for these proteins and discuss their roles in stress-responsive gene expression.
The phytohormone abscisic acid (ABA) plays an essential role in adaptive stress responses. The hormone regulates, among others, the expression of numerous stress-responsive genes. From various promoter analyses, ABA-responsive elements (ABREs) have been determined and a number of ABRE binding factors have been isolated, although their in vivo roles are not known. Here we report that the ABRE binding factors ABF3 and ABF4 function in ABA signaling. The constitutive overexpression of ABF3 or ABF4 in Arabidopsis resulted in ABA hypersensitivity and other ABA-associated phenotypes. In addition, the transgenic plants exhibited reduced transpiration and enhanced drought tolerance. At the molecular level, altered expression of ABA/stress-regulated genes was observed. Furthermore, the temporal and spatial expression patterns of ABF3 and ABF4 were consistent with their suggested roles. Thus, our results provide strong in vivo evidence that ABF3 and ABF4 mediate stress-responsive ABA signaling.
Arabidopsis ERF proteins such as DREB1, DREB2, and CBF1 bind to the dehydration-responsive element (DRE), which has the sequence TACCGACAT. Mutation analyses reveal that a central 5 bp CCGAC core of the DRE is the minimal sequence motif (designated as the DRE motif in this paper), to which the ERF domain fragment of CBF1 (CBF1-F) binds specifically with a binding K(d) at the nanomolar level. In contrast, the ERF domain fragment of the tobacco ERF2 (NtERF2-F) does not interact with the DRE motif, but restrictedly recognizes the sequence containing a minimal 6 bp GCCGCC motif (designated as the GCC motif in this paper). However, CBF1-F binds to the GCC motif with a binding activity similar to its binding activity for the DRE motif. These in vitro binding variations were further demonstrated through reporter cotransformation assays, suggesting that the DRE and GCC motifs are two similar sequence motifs sharing a common core region of CCGNC with a discriminating guanine base at the 5'-end of the GCC motif. Binding analyses with the mutated ERF domain show that such a unique binding of NtERF2-F to the GCC motif can be altered by the substitution of A14 with valine in beta-strand 2 of its ERF domain, the mutant NtERF2-F, ERFav, acquiring a binding to the DRE motif with a K(d) comparable to that for CBF1-F binding to the DRE motif. This demonstrates that A14 is an important determinant of the NtERF2-F binding specificity. A possible mechanism of the binding specificity determination is discussed.
Many plants, including Arabidopsis, increase in freezing tolerance in response to low, nonfreezing temperatures, a phenomenon known as cold acclimation. Previous studies established that cold acclimation involves rapid expression of the CBF transcriptional activators (also known as DREB1 proteins) in response to low temperature followed by induction of the CBF regulon (CBF-targeted genes), which contributes to an increase in freezing tolerance. Here, we present the results of transcriptome-profiling experiments indicating the existence of multiple low-temperature regulatory pathways in addition to the CBF cold response pathway. The transcript levels of approximately 8000 genes were determined at multiple times after plants were transferred from warm to cold temperature and in warm-grown plants that constitutively expressed CBF1, CBF2, or CBF3. A total of 306 genes were identified as being cold responsive, with transcripts for 218 genes increasing and those for 88 genes decreasing threefold or more at one or more time points during the 7-day experiment. These results indicate that extensive downregulation of gene expression occurs during cold acclimation. Of the cold-responsive genes, 48 encode known or putative transcription factors. Two of these, RAP2.1 and RAP2.6, were activated by CBF expression and thus presumably control subregulons of the CBF regulon. Transcriptome comparisons indicated that only 12% of the cold-responsive genes are certain members of the CBF regulon. Moreover, at least 28% of the cold-responsive genes were not regulated by the CBF transcription factors, including 15 encoding known or putative transcription factors, indicating that these cold-responsive genes are members of different low-temperature regulons. Significantly, CBF expression at warm temperatures repressed the expression of eight genes that also were downregulated by low temperature, indicating that in addition to gene induction, gene repression is likely to play an integral role in cold acclimation.
Transcriptional control of the expression of stress-responsive genes is a crucial part of the plant response to a range of abiotic and biotic stresses. Research carried out in the past few years has been productive in identifying transcription factors that are important for regulating plant responses to these stresses. These studies have also revealed some of the complexity and overlap in the responses to different stresses, and are likely to lead to new ways to enhance crop tolerance to disease and environmental stress.
Salt and drought stress signal transduction consists of ionic and osmotic homeostasis signaling pathways, detoxification (i.e., damage control and repair) response pathways, and pathways for growth regulation. The ionic aspect of salt stress is signaled via the SOS pathway where a calcium-responsive SOS3-SOS2 protein kinase complex controls the expression and activity of ion transporters such as SOS1. Osmotic stress activates several protein kinases including mitogen-activated kinases, which may mediate osmotic homeostasis and/or detoxification responses. A number of phospholipid systems are activated by osmotic stress, generating a diverse array of messenger molecules, some of which may function upstream of the osmotic stress-activated protein kinases. Abscisic acid biosynthesis is regulated by osmotic stress at multiple steps. Both ABA-dependent and -independent osmotic stress signaling first modify constitutively expressed transcription factors, leading to the expression of early response transcriptional activators, which then activate downstream stress tolerance effector genes.
Molecular and genomic studies have shown that several genes with various functions are induced by drought and cold stresses, and that various transcription factors are involved in the regulation of stress-inducible genes. The products of stress-inducible genes function not only in stress tolerance but also in stress response. Genetic studies have identified many factors that modify the regulation of stress responses. Recent progress has been made in analyzing the complex cascades of gene expression in drought and cold stress responses, especially in identifying specificity and crosstalk in stress signaling.
Casein kinases are critical in cell division and differentiation across species. A rice cDNA fragment encoding a putative casein kinase I (CKI) was identified via cDNA macroarray under brassinosteroid (BR) treatment, and a 1939-bp full-length cDNA, OsCKI1, was isolated and found to encode a putative 463-aa protein. RT-PCR and Northern blot analysis indicated that OsCKI1 was constitutively expressed in various rice tissues and upregulated by treatments with BR and abscisic acid (ABA). Enzymatic assay of recombinant OsCKI1 proteins expressed in Escherichia coli showed that the protein was capable of phosphorylating casein. The physiological roles of OsCKI1 were studied through antisense transgenic approaches, and homozygous transgenic plants showed abnormal root development, including fewer lateral and adventitious roots, and shortened primary roots as a result of reduced cell elongation. Treatment of wild-type plants with CKI-7, a specific inhibitor of CKI, also confirmed these functions of OsCKI1. Interestingly, in transgenic and CKI-7-treated plants, exogenously supplied IAA could restore normal root development, and measurement of free IAA content in CKI-deficient primary and adventitious roots revealed altered auxin content, indicating that OsCKI1 is involved in auxin metabolism or that it may affect auxin levels. Transgenic plants were less sensitive than control plants to ABA or BR treatment during germination, suggesting that OsCKI1 may be involved in various hormone-signaling pathways. OsCKI1-GFP fusion studies revealed the localization of OsCKI1 to the nucleus, suggesting a possible involvement in regulation of gene expression. In OsCKI1-deficient plants, differential gene expression was investigated using cDNA chip technology, and results indicated that genes related to signal transduction and hormone metabolism were indeed with altered expression.
Gram-negative bacteria use a variety of virulence factors including phytotoxins, exopolysaccharides, effectors secreted by the type III secretion system, and cell-wall-degrading enzymes to promote parasitism in plants. However, little is known about how these virulence factors alter plant cellular responses to promote disease. In this study, we show that virulent Pseudomonas syringae strains activate the transcription of an Arabidopsis ethylene response factor (ERF) gene, RAP2.6, in a coronatine insensitive 1 (COI1)-dependent manner. A highly sensitive RAP2.6 promoter-firefly luciferase (RAP2.6-LUC) reporter line was developed to monitor activities of various bacterial virulence genes. Analyses of P. syringae pv. tomato DC3000 mutants indicated that both type III secretion system and the phytotoxin coronatine are required for RAP2.6 induction. We show that at least five individual type III effectors, avirulence B (AvrB), AvrRpt2, AvrPphB, HopPtoK, and AvrPphEPto, contributed to RAP2.6 induction. Gene-for-gene recognition was not involved in RAP2.6 induction because plants lacking RPM1 and RPS2 responded normally to AvrB and AvrRpt2 in RAP2.6 expression. Interestingly, the role of coronatine in RAP2.6 induction can be partially substituted by the addition of avrB in DC3000, suggesting that AvrB may mimic coronatine. These results suggest that P. syringae type III effectors and coronatine act by augmenting a COI1-dependent pathway to promote parasitism.
Many plants increase in freezing tolerance upon exposure to low nonfreezing temperatures, a phenomenon known as cold acclimation. In this review, recent advances in determining the nature and function of genes with roles in freezing tolerance and the mechanisms involved in low temperature gene regulation and signal transduction are described. One of the important conclusions to emerge from these studies is that cold acclimation includes the expression of certain cold-induced genes that function to stabilize membranes against freeze-induced injury. In addition, a family of Arabidopsis transcription factors, the CBF/DREB1 proteins, have been identified that control the expression of a regulon of cold-induced genes that increase plant freezing tolerance. These results along with many of the others summarized here further our understanding of the basic mechanisms that plants have evolved to survive freezing temperatures. In addition, the findings have potential practical applications as freezing temperatures are a major factor limiting the geographical locations suitable for growing crop and horticultural plants and periodically account for significant losses in plant productivity.
Summary An efficient yeast-based system was developed for the isolation of plant cDNAs encoding transcription factors (TFs) and proteins with transcription activation functions (co-activators). The system consists of two vectors: (i) a reporter vector (pG221) harboring the iso-1-cytochrome c (CYC1) core promoter and the beta-galactosidase (lacZ) gene; and (ii) a cDNA library construction vector (pYF503), which yields a library of plant peptides fused to the GAL4-binding domain (GAL4-BD). Expression of a peptide harboring the characteristics of a transcriptional activator leads to expression of lacZ, allowing for selection of relevant colonies. TFs during rice embryo development were isolated through this system. Approximately 200 confirmed positive colonies were obtained from screening 10(6) yeast colonies, and sequence analysis of conserved domains identified 75 independent cDNAs, 20 of which encoded plant TFs or co-activators, including members of the APETALA2 (AP2)/ethylene-responsive element-binding protein (EREBP), MYB and growth-regulating factor (GRF) families. Peptides encoded by 13 of the isolated cDNAs were classified as potential TFs or co-activators because of the presence of conserved TF-like domains. Additionally, 2, 11, and 13 clones encoded kinases, chromosome-related proteins, and unknown proteins, respectively, while the remaining 16 cDNAs were associated with specific functions seemingly unrelated to TFs. Expression pattern analysis of selected TF-encoding genes via RT-PCR revealed that these genes were expressed during seed development, with differential transcription observed during various stages. This work provides informative hints for further study of the regulatory mechanism of rice seed development and illustrates an identification strategy that will be of practical value for the isolation of TFs and co-activators associated with specific plant developmental processes.