Overexpression of soybean DREB1 enhances drought stress tolerance of transgenic wheat in the field.

Drought-response-element binding (DREB)-like transcription factors can significantly enhance plant tolerance to water stress. However, most research on DREB-like proteins to date has been conducted in growth chambers or greenhouses, so there is very little evidence available to support their practical use in the field. In this study, we overexpressed GmDREB1 from soybean in two popular wheat varieties and conducted drought-tolerance experiments across a range of years, sites, and drought-stress regimes. We found that the transgenic plants consistently exhibited significant improvements in yield performance and a variety of physiological traits compared with wild-type plants when grown under limited water conditions in the field, for example showing grain yield increases between 4.79-18.43%. Specifically, we found that the transgenic plants had reduced membrane damage and enhanced osmotic adjustment and photosynthetic efficiency compared to the non-transgenic controls. Three enzymes from the biosynthetic pathway of the phytohormone melatonin were up-regulated in the transgenic plants, and external application of melatonin was found to improve drought tolerance. Together, our results demonstrate the utility of transgenic overexpression of GmDREB1 to improve the drought tolerance of wheat in the field.

Genome-wide analysis and identification of the low potassium stress responsive gene SiMYB3 in foxtail millet (Setariaitalica L.).

BACKGROUND: Potassium (K) is essential to plant growth and development. Foxtail millet (Setaria italic L.) is an important fodder grain crop in arid and semi-arid regions of Asia and Africa because of its strong tolerance to drought and barren stresses. The molecular mechanisms of physiological and biochemical responses and regulations to various abiotic stresses such as low potassium conditions in foxtail millet are not fully understood, which hinders the research and exploitation of this valuable resource. RESULTS: In this research, we demonstrated that the millet variety Longgu 25 was the most insensitive variety to low potassium stress among other five varieties. The transcriptome analysis of Longgu 25 variety revealed a total of 26,192 and 26,849 genes from the K+-deficient and normal transcriptomic libraries by RNA-seq, respectively. A total of 1982 differentially expressed genes (DEGs) were identified including 866 up-regulated genes and 1116 down-regulated genes. We conducted a comparative analysis of these DEGs under low-K+ stress conditions and discovered 248 common DEGs for potassium deprivation among foxtail millet, rice and Arabidopsis. Further Gene Ontology (GO) enrichment analysis identified a series of candidate genes that may involve in K+-deficient response and in intersection of molecular functions among foxtail millet, rice and Arabidopsis. The expression profiles of randomly selected 18 candidate genes were confirmed as true DEGs with RT-qPCR. Furthermore, one of the 18 DEGs, SiMYB3, is specifically expressed only in the millet under low-K+ stress conditions. Overexpression of SiMYB3 promoted the main root elongation and improved K+ deficiency tolerance in transgenic Arabidopsis plants. The fresh weight of the transgenic plants was higher, the primary root length was longer and the root surface-area was larger than those of control plants after K+ deficiency treatments. CONCLUSIONS: This study provides a global view of transcriptomic resources relevant to the K+-deficient tolerance in foxtail millet, and shows that SiMYB3 is a valuable genetic resource for the improvement of K+ deficiency tolerance in foxtail millet.

Genome-wide analysis of autophagy-associated genes in foxtail millet (Setaria italica L.) and characterization of the function of SiATG8a in conferring tolerance to nitrogen starvation in rice.

BACKGROUND: Autophagy is a cellular degradation process that is highly evolutionarily-conserved in yeast, plants, and animals. In plants, autophagy plays important roles in regulating intracellular degradation and recycling of amino acids in response to nutrient starvation, senescence, and other environmental stresses. Foxtail millet (Setaria italica) has strong resistance to stresses and has been proposed as an ideal material for use in the study of the physiological mechanisms of abiotic stress tolerance in plants. Although the genome sequence of foxtail millet (Setaria italica) is available, the characteristics and functions of abiotic stress-related genes remain largely unknown for this species. RESULTS: A total of 37 putative ATG (autophagy-associated genes) genes in the foxtail millet genome were identified. Gene duplication analysis revealed that both segmental and tandem duplication events have played significant roles in the expansion of the ATG gene family in foxtail millet. Comparative synteny mapping between the genomes of foxtail millet and rice suggested that the ATG genes in both species have common ancestors, as their ATG genes were primarily located in similar syntenic regions. Gene expression analysis revealed the induced expression of 31 SiATG genes by one or more phytohormone treatments, 26 SiATG genes by drought, salt and cold, 24 SiATG genes by darkness and 25 SiATG genes by nitrogen starvation. Results of qRT-PCR showing that among 37 SiATG genes, the expression level of SiATG8a was the highest after nitrogen starvation treatment 24 h, suggesting its potential role in tolerance to nutrient starvation. Moreover, the heterologous expression of SiATG8a in rice improved nitrogen starvation tolerance. Compared to wild type rice, the transgenic rice performed better and had higher aboveground total nitrogen content when the plants were grown under nitrogen starvation conditions. CONCLUSIONS: Our results deepen understanding about the characteristics and functions of ATG genes in foxtail millet and also identify promising new genetic resources that should be of use in future efforts to develop varieties of foxtail millet and other crop species that have resistance to nitrogen deficiency stress.

An iNTT system for the large-scale screening of differentially expressed, nuclear-targeted proteins: cold-treatment-induced nucleoproteins in Rye (Secale cereale L.).

BACKGROUND: Nuclear proteins play critical roles in regulating mRNA transcription and processing, DNA replication, and epigenetic genome modification. Therefore, the ability to monitor changes in nuclear proteins is helpful not only to identify important regulatory proteins but also to study the mechanisms of actions of nuclear proteins. However, no effective methods have been developed yet. Rye is strongly resistant to various biotic and abiotic stresses; however, few genes have been functionally characterized to date due to the complexity of its genome and a lack of genomic sequence information. RESULTS: We developed an integrative Nuclear Transportation Trap (iNTT) system that includes an improved nuclear transportation trap and utilizes the "after suppression subtraction" method. Oligonucleotides encoding a nuclear localization signal (NLS) or a transcription factor, GmAREB, were inserted into pLexAD or pLexAD-NES, respectively, and then transformed into yeast cells (EGY48). We showed that the pLexAD vector expressing a cDNA library in the iNTT system was more efficient for screening than the vector pLexAD-NES, which has previously been used in an NTT system. We used the iNTT system to screen a cDNA library of cold-treated rye. A total of 241 unique genes were identified, including 169 differentially expressed proteins; of these, 106 were of known and 63 were of unknown function. Moreover, 82 genes (49 %) among the 169 differentially expressed genes were predicted to contain an NLS domain. Thirty-three (31 %) of the 106 functionally known proteins have DNA-binding activity. To test the specificity of the nuclear proteins identified using the iNTT screen, four of the proteins differentially expressed in response to temperature stress, ScT1 (a heat shock protein), ScT36 (a MYB-like transcription factor), ScT133 (an ERF-like transcription factor) and ScT196 (a protein of unknown function), were studied in more depth. These proteins were shown to exclusively localize to the nucleus, and their expression levels were increased in response to low-temperature stress. To identify the function of these screened nuclear proteins, ScT1- and ScT36-transgenic Arabidopsis plants were constructed, and ScT1 or ScT36 overexpression was found to enhance tolerance to high-temperature or freezing stresses, respectively. CONCLUSIONS: The newly developed iNTT system provides an effective method for identifying nuclear-targeted proteins and monitoring induced expression levels. ScT1 and ScT36 might be good candidate genes for improving the stress tolerance of plants by genetic transformation.

Investigation of the ASR family in foxtail millet and the role of ASR1 in drought/oxidative stress tolerance.

KEY MESSAGE: Six foxtail millet ASR genes were regulated by various stress-related signals. Overexpression of ASR1 increased drought and oxidative tolerance by controlling ROS homeostasis and regulating oxidation-related genes in tobacco plants. Abscisic acid stress ripening (ASR) proteins with ABA/WDS domains constituted a class of plant-specific transcription factors, playing important roles in plant development, growth and abiotic stress responses. However, only a few ASRs genes have been characterized in crop plants and none was reported so far in foxtail millet (Setaria italic), an important drought-tolerant crop and model bioenergy grain crop. In the present study, we identified six foxtail millet ASR genes. Gene structure, protein alignments and phylogenetic relationships were analyzed. Transcript expression patterns of ASR genes revealed that ASRs might play important roles in stress-related signaling and abiotic stress responses in diverse tissues in foxtail millet. Subcellular localization assays showed that SiASR1 localized in the nucleus. Overexpression of SiASR1 in tobacco remarkably increased tolerance to drought and oxidative stresses, as determined through developmental and physiological analyses of germination rate, root growth, survival rate, relative water content, ion leakage, chlorophyll content and antioxidant enzyme activities. Furthermore, expression of SiASR1 modulated the transcript levels of oxidation-related genes, including NtSOD, NtAPX, NtCAT, NtRbohA and NtRbohB, under drought and oxidative stress conditions. These results provide a foundation for evolutionary and functional characterization of the ASR gene family in foxtail millet.

AtTGA4, a bZIP transcription factor, confers drought resistance by enhancing nitrate transport and assimilation in Arabidopsis thaliana.

To cope with environmental stress caused by global climate change and excessive nitrogen application, it is important to improve water and nitrogen use efficiencies in crop plants. It has been reported that higher nitrogen uptake could alleviate the damaging impact of drought stress. However, there is scant evidence to explain how nitrogen uptake affects drought resistance. In this study we observed that bZIP transcription factor AtTGA4 (TGACG motif-binding factor 4) was induced by both drought and low nitrogen stresses, and that overexpression of AtTGA4 simultaneously improved drought resistance and reduced nitrogen starvation in Arabidopsis. Following drought stress there were higher nitrogen and proline contents in transgenic AtTGA4 plants than in wild type controls, and activity of the key enzyme nitrite reductase (NIR) involved in nitrate assimilation processes was also higher. Expressions of the high-affinity nitrate transporter genes NRT2.1 and NRT2.2 and nitrate reductase genes NIA1 and NIA2 in transgenic plants were all higher than in wild type indicating that higher levels of nitrate transport and assimilation activity contributed to enhanced drought resistance of AtTGA4 transgenic plants. Thus genetic transformation with AtTGA4 may provide a new approach to simultaneously improve crop tolerance to drought and low nitrogen stresses.

Overexpression of the autophagy-related gene SiATG8a from foxtail millet (Setaria italica L.) confers tolerance to both nitrogen starvation and drought stress in Arabidopsis.

Autophagy is an evolutionarily conserved biological process in all eukaryotes for the degradation of intracellular components for nutrient recycling. Autophagy is known to be involved in responses to low nitrogen stress in Arabidopsis. Foxtail millet has strong abiotic stress resistance to both low nutrient and drought stress. However, to date, there have only been a few genes reported to be related with abiotic stress resistance in foxtail millet. In this study, we identified an autophagy-related gene, SiATG8a, from foxtail millet. SiATG8a is mainly expressed in stems and its expression was dramatically induced by drought stress and nitrogen starvation treatments. SiATG8a was localized in the membrane and cytoplasm of foxtail millet. Overexpression of SiATG8a in Arabidopsis conferred tolerance to both nitrogen starvation and to drought stress. Under nitrogen starvation conditions, the SiATG8a transgenic plants had larger root and leaf areas and accumulated more total nitrogen than wild-type plants. The transgenic plants had lower total protein concentrations than did the WT plants. Under drought stress, the SiATG8a transgenic plants had higher survival rates, chlorophyll content, and proline content, but had lower MDA content than wild type plants. Taken together, our results represent the first identified case where overexpression of autophagy related gene can simultaneously improve plant resistance to low nitrogen and drought stresses. These findings implicate plant autophagy in plant stress responses to low nitrogen and drought and should be helpful in efforts to improve stresses resistance to nitrogen starvation and drought of crops by genetic transformation.

ABI-like transcription factor gene TaABL1 from wheat improves multiple abiotic stress tolerances in transgenic plants.

The phytohormone abscisic acid (ABA) plays crucial roles in adaptive responses of plants to abiotic stresses. ABA-responsive element binding proteins (AREBs) are basic leucine zipper transcription factors that regulate the expression of downstream genes containing ABA-responsive elements (ABREs) in promoter regions. A novel ABI-like (ABA-insensitive) transcription factor gene, named TaABL1, containing a conserved basic leucine zipper (bZIP) domain was cloned from wheat. Southern blotting showed that three copies were present in the wheat genome. Phylogenetic analyses indicated that TaABL1 belonged to the AREB subfamily of the bZIP transcription factor family and was most closely related to ZmABI5 in maize and OsAREB2 in rice. Expression of TaABL1 was highly induced in wheat roots, stems, and leaves by ABA, drought, high salt, and low temperature stresses. TaABL1 was localized inside the nuclei of transformed wheat mesophyll protoplast. Overexpression of TaABL1 enhanced responses of transgenic plants to ABA and hastened stomatal closure under stress, thereby improving tolerance to multiple abiotic stresses. Furthermore, overexpression of TaABL1 upregulated or downregulated the expression of some stress-related genes controlling stomatal closure in transgenic plants under ABA and drought stress conditions, suggesting that TaABL1 might be a valuable genetic resource for transgenic molecular breeding.

Durable field resistance to wheat yellow mosaic virus in transgenic wheat containing the antisense virus polymerase gene.

Wheat yellow mosaic virus (WYMV) has spread rapidly and causes serious yield losses in the major wheat-growing areas in China. Because it is vectored by the fungus-like organism Polymyxa graminis that survives for long periods in soil, it is difficult to eliminate by conventional crop management or fungicides. There is also only limited resistance in commercial cultivars. In this research, fourteen independent transgenic events were obtained by co-transformation with the antisense NIb8 gene (the NIb replicase of WYMV) and a selectable gene bar. Four original transgenic lines (N12, N13, N14 and N15) and an offspring line (N12-1) showed high and durable resistance to WYMV in the field. Four resistant lines were shown to have segregated and only contain NIb8 (without bar) by PCR and herbicide resistance testing in the later generations. Line N12-1 showed broad-spectrum resistance to WYMV isolates from different sites in China. After growing in the infested soil, WYMV could not be detected by tissue printing and Western blot assays of transgenic wheat. The grain yield of transgenic wheat was about 10% greater than the wild-type susceptible control. Northern blot and small RNA deep sequencing analyses showed that there was no accumulation of small interfering RNAs targeting the NIb8 gene in transgenic wheat plants, suggesting that transgene RNA silencing, a common mechanism of virus-derived disease resistance, is not involved in the process of WYMV resistance. This durable and broad-spectrum resistance to WYMV in transgenic wheat will be useful for alleviating the damage caused by WYMV.

Study on Shear Performance of Corroded Steel Fiber Reinforced Concrete Beams under Impact Load.

With the growing use of steel-fiber-reinforced-concrete (SFRC) beams in environmentally friendly and rapid construction, it is essential to assess their impact performance. These beams may encounter unexpected impact loadings from accidents or terrorist attacks during service life. This study explored the impact of steel fiber content and drop hammer height on the impact load testing of corrosion-treated SFRC beams. Experiments were conducted with varying steel fiber contents (0%, 0.25%, 0.5%, 0.75%, and 1.0%), and drop hammer height (1 m, 2 m, and 3 m). The corrosion test demonstrates that SFRC beams supplemented with steel fibers showcase a diminished surface rust spot area in comparison to those lacking fibers. This improvement is ascribed to the bonding between fibers and the concrete matrix, along with their current-sharing properties. SFRC beams, subjected to impact testing, exhibit concrete crushing at the top without spalling, showcasing improved impact resistance due to increased fiber content, which reduces crack formation. Additionally, different fiber contents yield varied responses to impact loads, with higher fiber content notably enhancing overall beam performance and energy dissipation capacity. Energy dissipation analysis shows a moderate increase with higher fiber contents, and impulse impact force generally rises with fiber content, indicating improved impact resistance.

IL-35 Stabilizes Treg Phenotype to Protect Cardiac Allografts in Mice.

BACKGROUND: Interleukin-35 (IL-35), secreted by regulatory T cells (Treg) and B cells, is immunosuppressive under both physiological and pathological conditions. However, the role of IL-35 in all responses has yet to be investigated. Here, we demonstrate that IL-35 protects allografts by stabilizing the Treg phenotype and suppressing CD8 + T-cell activation in a mouse heart transplantation model. METHODS: The effect of IL-35 on immune cell infiltration in grafts and secondary lymphoid organs was examined using mass cytometry, flow cytometry, and immunofluorescence. Moreover, using quantitative real-time polymerase chain reaction, flow cytometry, and phospho-flow assays, we demonstrated that IL-35 maintains Treg phenotypes to restrain CD8 + T cells via the gp130/signal transducer and activator of transcription 1 pathway. RESULTS: Mass cytometry analysis of intragraft immune cells showed that IL-35 decreased CD8 + T-cell infiltration and increased Foxp3 and IL-35 expressions in Treg. In vitro, we demonstrated that IL-35 directly promoted Treg phenotypic and functional stability and its IL-35 secretion, generating a positive feedback loop. However, Treg are required for IL-35 to exert its suppressive effect on CD8 + T cells in vitro. After depleting Treg in the recipient, IL-35 did not prolong graft survival or decrease CD8 + T-cell infiltration. Mechanistically, we found that IL-35 sustained Treg stability via the gp130/signal transducer and activator of transcription 1 signaling pathway. CONCLUSIONS: Our findings highlight that IL-35 stabilizes the Treg phenotype to ameliorate CD8 + T-cell infiltration in the allograft, which has never been described in the transplanted immunological milieu.

A wheat PI4K gene whose product possesses threonine autophophorylation activity confers tolerance to drought and salt in Arabidopsis.

Phosphoinositides are involved in regulation of recruitment and activity of signalling proteins in cell membranes. Phosphatidylinositol (PI) 4-kinases (PI4Ks) generate PI4-phosphate the precursor of regulatory phosphoinositides. No type II PI4K research on the abiotic stress response has previously been reported in plants. A stress-inducible type II PI4K gene, named TaPI4KIIγ, was obtained by de novo transcriptome sequencing of drought-treated wheat (Triticum aestivum). TaPI4KIIγ, localized on the plasma membrane, underwent threonine autophosphorylation, but had no detectable lipid kinase activity. Interaction of TaPI4KIIγ with wheat ubiquitin fusion degradation protein (TaUDF1) indicated that it might be hydrolysed by the proteinase system. Overexpression of TaPI4KIIγ revealed that it could enhance drought and salt stress tolerance during seed germination and seedling growth. A ubdkγ7 mutant, identified as an orthologue of TaPI4KIIγ in Arabidopsis, was sensitive to salt, polyethylene glycol (PEG), and abscisic acid (ABA), and overexpression of TaPI4KIIγ in the ubdkγ7 mutant compensated stress sensitivity. TaPI4KIIγ promoted root growth in Arabidopsis, suggesting that TaPI4KIIγ might enhance stress resistance by improving root growth. Overexpression of TaPI4KIIγ led to an altered expression level of stress-related genes and changes in several physiological traits that made the plants more tolerant to stress. The results provided evidence that overexpression of TaPI4KIIγ could improve drought and salt tolerance.

The voltage-dependent anion channel 1 (AtVDAC1) negatively regulates plant cold responses during germination and seedling development in Arabidopsis and interacts with calcium sensor CBL1.

The voltage-dependent anion channel (VDAC), a highly conserved major mitochondrial outer membrane protein, plays crucial roles in energy metabolism and metabolite transport. However, knowledge about the roles of the VDAC family in plants is limited. In this study, we investigated the expression pattern of VDAC1 in Arabidopsis and found that cold stress promoted the accumulation of VDAC1 transcripts in imbibed seeds and mature plants. Overexpression of VDAC1 reduced tolerance to cold stress in Arabidopsis. Phenotype analysis of VDAC1 T-DNA insertion mutant plants indicated that a vdac1 mutant line had faster germination kinetics under cold treatment and showed enhanced tolerance to freezing. The yeast two-hybrid system revealed that VDAC1 interacts with CBL1, a calcium sensor in plants. Like the vdac1, a cbl1 mutant also exhibited a higher seed germination rate. We conclude that both VDAC1 and CBL1 regulate cold stress responses during seed germination and plant development.

A mutation in Arabidopsis BSK5 encoding a brassinosteroid-signaling kinase protein affects responses to salinity and abscisic acid.

As the most recently characterized group of plant hormones, brassinosteroids (BR) are involved in a number of physiological responses. Although many key components of the BR signaling pathway have been isolated and characterized, there is little information on detailed characterization of brassinosteroid-signaling kinase (BSK) proteins. In this study, Arabidopsis BSK5 was isolated and functionally analyzed. BSK5 transcripts were detected in various tissues, and were induced by abiotic stresses including salt and drought, as well as phytohormones of BR and abscisic acid (ABA). Arabidopsis loss-of-function mutant bsk5 exhibited sensitivity to salinity and ABA. Mutations of the BSK5 gene also altered the expression of several stress-regulated genes. We suggest that BSK5 responds to other signals as well as BR.

Overexpression of soybean GmCBL1 enhances abiotic stress tolerance and promotes hypocotyl elongation in Arabidopsis.

Although extensive studies and remarkable progress have been made with Arabidopsis calcineurin B-like proteins (CBLs), knowledge of their functions in other plant species is still limited. Here we isolated gene GmCBL1 from soybean, a homolog of AtCBL1 in Arabidopsis. GmCBL1 was differentially induced by multiple abiotic stress and plant hormones, and its transcripts were abundant in seedlings and mature roots. We over-expressed GmCBL1 in Arabidopsis and found that it enhanced tolerances to both high salt and drought stresses in the transgenic plants. Overexpression of GmCBL1 also promoted hypocotyl elongation under light conditions. GmCBL1 may regulate stress tolerance through activation of stress-related genes, and may control hypocotyl development by altering the expression of gibberellin biosynthesis-related genes. This study identifies a putative soybean CBL gene that functions in both stress tolerance and light-dependent hypocotyl development.

Heat shock protein 90 in plants: molecular mechanisms and roles in stress responses.

The heat shock protein 90 (Hsp90) family mediates stress signal transduction, and plays important roles in the control of normal growth of human cells and in promoting development of tumor cells. Hsp90s have become a currently important subject in cellular immunity, signal transduction, and anti-cancer research. Studies on the physiological functions of Hsp90s began much later in plants than in animals and fungi. Significant progress has been made in understanding complex mechanisms of HSP90s in plants, including ATPase-coupled conformational changes and interactions with cochaperone proteins. A wide range of signaling proteins interact with HSP90s. Recent studies revealed that plant Hsp90s are important in plant development, environmental stress response, and disease and pest resistance. In this study, the plant HSP90 family was classified into three clusters on the basis of phylogenetic relationships, gene structure, and biological functions. We discuss the molecular functions of Hsp90s, and systematically review recent progress of Hsp90 research in plants.

The soybean GmbZIP1 transcription factor enhances multiple abiotic stress tolerances in transgenic plants.

Abscisic acid (ABA)-responsive element binding proteins (AREBs) are basic domain/leucine zipper transcription factors that bind to the ABA-responsive element (ABRE) in the promoter regions of ABA-inducible genes in plants. A novel bZIP transcription factor gene, GmbZIP1, encoding 438 amino acids with a conserved bZIP domain composed of 60 amino acids was isolated from salt-tolerant soybean cv. Tiefeng 8. Southern blotting showed that only one copy was present in the soybean genome. Phylogenetic analyses showed that GmbZIP1 belonged to the AREB subfamily of the bZIP family and was most closely related to AtABF2 and OsTRAB1. The expression of GmbZIP1 was highly induced by ABA, drought, high salt and low temperature; and GmbZIP1 was expressed in soybean roots, stems and leaves under different stress conditions. GmbZIP1 was localized inside the nuclei of transformed onion epidermal cells. Overexpression of GmbZIP1 enhanced the responses of transgenic plants to ABA and triggered stomatal closure under stresses, potentially leading to improved tolerances to several abiotic stresses such as high salt, low temperature and drought in transgenic plants. Furthermore, overexpression of GmbZIP1 affected the expression of some ABA or stress-related genes involved in regulating stomatal closure in Arabidopsis under ABA, drought and high salt stress conditions. A few AREB elements were detected in the promoter region of those ABA or stress-related genes, suggesting that GmbZIP1 regulates the ABA response or stomatal closure mediated by those downstream genes in transgenic Arabidopsis. Moreover, GmbZIP1 was used to improve the drought tolerance trait of Chinese wheat varieties BS93. Functional analysis showed that overexpression of GmbZIP1 enhanced the drought tolerance of transgenic wheat, and transcripts of GmbZIP1 were detected in transgenic wheat using RT-PCR. In addition, GmbZIP1 overexpression did not result in growth retardation in all transgenic plants, suggesting that GmbZIP1 may be a valuable genetic resource for engineering stress tolerance of crops.

Functions and application of the AP2/ERF transcription factor family in crop improvement.

Plants have acquired sophisticated stress response systems to adapt to changing environments. It is important to understand plants' stress response mechanisms in the effort to improve crop productivity under stressful conditions. The AP2/ERF transcription factors are known to regulate diverse processes of plant development and stress responses. In this study, the molecular characteristics and biological functions of AP2/ERFs in a variety of plant species were analyzed. AP2/ERFs, especially those in DREB and ERF subfamilies, are ideal candidates for crop improvement because their overexpression enhances tolerances to drought, salt, freezing, as well as resistances to multiple diseases in the transgenic plants. The comprehensive analysis of physiological functions is useful in elucidating the biological roles of AP2/ERF family genes in gene interaction, pathway regulation, and defense response under stress environments, which should provide new opportunities for the crop tolerance engineering.

Isolation and characterization of a novel EAR-motif-containing gene GmERF4 from soybean (Glycine max L.).

Transcriptional repressors are emerging as central regulators of development and stress responses in different organisms. The ERF-associated amphiphilic repression (EAR) motif was identified as essential for transcriptional repression. To gain a better understanding of this type of protein, we reported here a novel GmERF4 protein from soybean. Sequence alignment showed that GmERF4 contains one AP2/ERF domain, two putative nuclear localization signal regions and one EAR motif. The GmERF4 protein was preferentially localized to the nucleus of onion epidermis cells and bound specifically to the GCC box and DRE/CRT element in vitro. Furthermore, the expression of GmERF4 was induced by ethylene, JA, SA, cold, salt, drought, and soybean mosaic virus, and repressed by ABA. Constitutive expression of GmERF4 in transgenic tobacco plants increased tolerance to salt and drought stresses compared with wild-type plants, but did not exhibit detectable resistance against bacterial infection.

Establishment and application of a novel fluorescence-based analytical method for the rapid detection of viable bacteria in different samples.

A rapid method for readily detecting the total numbers of viable bacterial cells in numerous samples (including surface water, solid inoculants, and soil samples) is reported using a newly developed hand-held fluorometer and a fluorescent dye Calcein UltraGreen™ AM. Compared to the traditional plate counting method that requires 48 hours of cultivation, the newly established method does not require any incubation time, making the detection method faster and more convenient. The portable rapid detection fluorometer has a wide dynamic range of relative fluorescence intensity from 45 to 30 133. It can detect bacterial concentration ranging from 105 to 1010 cells per mL. This newly established method has good applicability for accurately and quickly detecting the cell number of viable bacteria in various samples. The results of the fluorescence-based method were compared with those of the traditional plate counting method, and it was found that the relative standard deviation was less than 6%. This new rapid measurement system provides a robust method for the rapid on-site detection of viable bacteria.