ERFs (ethylene-responsive element binding factors) belong to a large family of plant transcription factors that are found exclusively in plants. A small subfamily of ERF proteins can act as transcriptional repressors. The Arabidopsis genome contains eight ERF repressors, namely AtERF3, AtERF4, and AtERF7 to AtERF12. Members of ERF repressors show differential expression, suggesting that they may have different function. Using a transient expression system, we demonstrated that AtERF4, AtERF7, AtERF10, AtERF11 and AtERF12 can function as transcriptional repressors. The expression of AtERF4 can be induced by ethylene, jasmonic acid, and abscisic acid (ABA). By using green fluorescent protein fusion, we demonstrated that AtEFR4 accumulated in the nuclear bodies of Arabidopsis cells. Expression of 35S:AtERF4-GFP in transgenic Arabidopsis plants conferred an ethylene-insensitive phenotype and repressed the expression of Basic Chitinase and beta-1,3-Glucanase, the GCC-box-containing genes. In comparison with wild-type plants, 35S:AtERF4-GFP transgenic plants had decreased sensitivity to ABA and were hypersensitive to sodium chloride. The expression of the ABA responsive genes, ABI2, rd29B and rab18, was decreased in the 35S:AtERF4-GFP transgenic plants. Our study provides evidence that AtERF4 is a negative regulator capable of modulating ethylene and abscisic acid responses.
SummaryHD2 (histone deacetylase) proteins are plant-speci®c histone deacetylases (HDACs). The Arabidopsis genome contains four HD2 genes, namely HD2A, HD2B, HD2C, and HD2D. We have previously demonstrated that HD2A, HD2B, and HD2C can repress transcription directly by targeting to promoters in planta. Here, we show that the N-terminal conserved motif (EFWG) and histidine 25 (H25), a potential catalytic residue, were important for the gene repression activity of HD2A. In situ hybridization indicated that HD2A, HD2B, and HD2C were expressed in ovules, embryos, shoot apical meristems, and primary leaves. Furthermore, all three genes were strongly induced during the process of somatic embryogenesis. HD2D mRNA was only detected in the stems and¯owers with young siliques and may have adopted different functions. Using green¯uorescent protein (GFP) fusions, we demonstrated that HD2A, HD2B, and HD2C accumulated in the nuclei of Arabidopsis cells. Overexpression of 35S::GFP±HD2A in transgenic Arabidopsis plants generated pleiotropic developmental abnormalities, including abnormal leaves, delayed¯owering, and aborted seed development. The data showed that normal pattern of HD2 expression was essential for normal plant development and that HD2A, HD2B, and HD2C may be needed for embryogenesis and embryo development. Reverse transcriptase (RT)-PCR analysis revealed that a number of genes involved in seed development and maturation were repressed in the 35S::GFP±HD2A plants, supporting a role of HD2A in the regulation of gene expression during seed development.
BackgroundAlfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production.ResultsIn this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line.ConclusionModifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines.
Chronic ingestion of the highly active, specific cysteine proteinase inhibitor, E‐64, has a profound effect on Colorado potato beetle (CPB) larval growth, development and survival, as well as on adult fecundity. However, the number of insects surviving to the adult stage did not decrease below 26% with increasing E‐64 concentration above 1.5 μg E‐64 cm−2 leaf surface. The development time to the pupal stage was increased from 13 days, when larvae were reared on control leaves, to 21 days at a concentration of 1.5 μg E‐64 cm−2. The most significant effect of dietary E‐64 was on adult fecundity, with mated females reared on untreated leaves laying an average 62 ± 5.7 eggs daily in the first 10 days, and those maintained on 0.5 μg E‐64 cm−2, laying only 16 ± 2.4 eggs day−1. Females given 1 μg E‐64 cm−2 laid few if any eggs, but started producing egg masses as large as control insects about 5 days after being switched to control leaves. These effects on the insect life cycle were directly related to the degree of inhibition of cysteine proteinase activity in gut extracts. The general proteinase activity in control extracts was 6.5 ± 0.16 units min−1 mg gut−1, which decreased to 1.9 ± 0.16 in guts of insects reared on 1 μg E‐64 cm−2. The proportion of proteinase activity inhibitable by E‐64 decreased from 66% in control guts to 10‐15% in guts from larvae reared on 1 μg E‐64 cm−2. The aspartate proteinase inhibitor, pepstatin, decreased proteinase activity by 35% in control guts. There was no induction of pepstatin‐inhibitable proteinases in response to inhibition by E‐64, and no inhibition of gut enzyme activity by soybean trypsin inhibitor from larvae fed any of the E‐64 concentrations. This study demonstrates that proteinase levels must be significantly reduced to have a pronounced effect on larval growth and survival, while fecundity of mated females is affected by lower concentrations of inhibitor. It also suggests that the CPB may be a difficult pest to control using a more specific, plant‐derived cysteine proteinase inhibitor, such as oryzacystatin.
Our previous studies have shown that tCUP, a cryptic promoter from tobacco, functions in all living plant cell types in a wide range of plant species. This led us to investigate if an enhanced derivative, EntCUP∆, could be used to drive the neomycin phosphotransferase II (nptII) gene and select for kanamycin resistance in crop species that regenerate by organogenesis or embryogenesis. Tobacco (leaves), cauliflower (hypocotyls) and alfalfa (leaves, petioles, stems) explants were co-cultivated with Agrobacterium containing either EntCUP∆-nptII-nos or 35S-nptII-nos to compare the efficiency of selection for kanamycin resistance. The infected alfalfa explants were placed in somatic embryo induction media, whereas tobacco and cauliflower explants were placed in shoot induction media with kanamycin at concentrations that normally inhibit regeneration. Transgenic plants were recovered from all of the explants with both selectable marker gene constructs. The transformation efficiencies using tCUP∆-nptII-nos were comparable to or higher than those using 35S-nptII-nos in all three species tested. This study demonstrated that promoters which are not associated with expressed plant genes can be used as alternatives for the expression of selectable marker genes in a broad range of tissues and species for the generation of transgenic plants.
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