Overexpression of the Arabidopsis 10-kilodalton acyl-coenzyme A-binding protein ACBP6 enhances freezing tolerance

Small 10-kD acyl-coenzyme A-binding proteins (ACBPs) are highly conserved proteins that are prevalent in eukaryotes. In Arabidopsis (Arabidopsis thaliana), other than the 10-kD ACBP homolog (designated Arabidopsis ACBP6), there are five larger forms of ACBPs ranging from 37.5 to 73.1 kD. In this study, the cytosolic subcellular localization of Arabidopsis ACBP6 was confirmed by analyses of transgenic Arabidopsis expressing autofluorescence-tagged ACBP6 and western-blot analysis of subcellular fractions using ACBP6-specific antibodies. The expression of Arabidopsis ACBP6 was noticeably induced at 48 h after 4 degrees C treatment by northern-blot analysis and western-blot analysis. Furthermore, an acbp6 T-DNA insertional mutant that lacked ACBP6 mRNA and protein displayed increased sensitivity to freezing temperature (-8 degrees C), while ACBP6-overexpressing transgenic Arabidopsis plants were conferred enhanced freezing tolerance. Northern-blot analysis indicated that ACBP6-associated freezing tolerance was not dependent on the induction of cold-regulated COLD-RESPONSIVE gene expression. Instead, ACBP6 overexpressors showed increased expression of mRNA encoding phospholipase Ddelta. Lipid profiling analyses of rosettes from cold-acclimated, freezing-treated (-8 degrees C) transgenic Arabidopsis plants overexpressing ACBP6 showed a decline in phosphatidylcholine (-36% and -46%) and an elevation of phosphatidic acid (73% and 67%) in comparison with wild-type plants. From our comparison, the gain in freezing tolerance in ACBP6 overexpressors that was accompanied by decreases in phosphatidylcholine and an accumulation of phosphatidic acid is consistent with previous findings on phospholipase Ddelta-overexpressing transgenic Arabidopsis. In vitro filter-binding assays indicating that histidine-tagged ACBP6 binds phosphatidylcholine, but not phosphatidic acid or lysophosphatidylcholine, further imply a role for ACBP6 in phospholipid metabolism in Arabidopsis, including the possibility of ACBP6 in the cytosolic trafficking of phosphatidylcholine.     

Overexpression of MtCAS31 enhances drought tolerance in transgenic Arabidopsis by reducing stomatal density

? Dehydrins are a type of late embryogenesis abundant protein. Some dehydrins are involved in the response to various abiotic stresses. Accumulation of dehydrins enhances the drought, cold and salt tolerances of transgenic plants, although the underlying mechanism is unclear. MtCAS31 (Medicago Truncatula cold-acclimation specific protein 31) is a Y(2)K(4)-type dehydrin that was isolated from Medicago truncatula. ? We analyzed the subcellular and histochemical localization of MtCAS31, and the expression patterns of MtCAS31 under different stresses. Transgenic Arabidopsis that overexpressed MtCAS31 was used to determine the function of MtCAS31. A yeast two-hybrid assay was used to screen potential proteins that could interact with MtCAS31. The interaction was confirmed by bimolecular fluorescence complementation (BiFC) assay. ? After a 3-h drought treatment, the expression of MtCAS31 significantly increased 600-fold. MtCAS31 overexpression dramatically reduced stomatal density and markedly enhanced the drought tolerance of transgenic Arabidopsis. MtCAS31 could interact with AtICE1 (inducer of CBF expression 1) and the AtICE1 homologous protein Mt7g083900.1, which was identified from Medicago truncatula both in vitro and in vivo. ? Our findings demonstrate that a dehydrin induces decreased stomatal density. Most importantly, the interaction of MtCAS31 with AtICE1 plays a role in stomatal development. We hypothesize that the interaction of MtCAS31 and AtICE1 caused the decrease in stomatal density to enhance the drought resistance of transgenic Arabidopsis.     

Overexpression of membrane-associated acyl-CoA-binding protein ACBP1 enhances lead tolerance in Arabidopsis

In Arabidopsis thaliana , a family of six genes encodes acyl-CoA-binding proteins (ACBPs) that show conservation at the acyl-CoA-binding domain. They are the membrane-associated ACBP1 and ACBP2, extracellularly targeted ACBP3, kelch-motif-containing ACBP4 and ACBP5, and 10-kDa ACBP6. The acyl-CoA domain in each of ACBP1 to ACBP6 binds long-chain acyl-CoA esters in vitro , suggestive of possible roles in plant lipid metabolism. We addressed here the use of Arabidopsis ACBPs in conferring lead [Pb(II)] tolerance in transgenic plants because the 10-kDa human ACBP has been identified as a molecular target for Pb(II) in vivo . We investigated the effect of Pb(II) stress on the expression of genes encoding Arabidopsis ACBP1, ACBP2 and ACBP6. We showed that the expression of ACBP1 and ACBP2 , but not ACBP6 , in root is induced by Pb(II) nitrate treatment. In vitro Pb(II)-binding assays indicated that ACBP1 binds Pb(II) comparatively better, and ACBP1 was therefore selected for further investigations. When grown on Pb(II)-containing medium, transgenic Arabidopsis lines overexpressing ACBP1 were more tolerant to Pb(II)-induced stress than the wild type. Accumulation of Pb(II) in shoots of the ACBP1 -overepxressing plants was significantly higher than wild type. The acbp1 mutant showed enhanced sensitivity to Pb(II) when germinated and grown in the presence of Pb(II) nitrate and tolerance was restored upon complementation using an ACBP1 cDNA. Our results suggest that ACBP1 is involved in mediating Pb(II) tolerance in Arabidopsis with accumulation of Pb(II) in shoots. Such observations of Pb(II) accumulation, rather than Pb(II) extrusion, in the ACBP1 -overexpressing plants implicate possible use of ACBP1 in Pb(II) phytoremediation.     

Overexpression of cinnamyl alcohol dehydrogenase gene from sweetpotato enhances oxidative stress tolerance in transgenic Arabidopsis

Cinnamyl alcohol dehydrogenase (CAD) is the enzyme in the last step of lignin biosynthetic pathway and is involved in the generation of lignin monomers. IbCAD1 gene in sweetpotato (Ipomoea batatas) was identified, and its expression was induced by abiotic stresses based on promoter analysis. In this study, transgenic Arabidopsis plants overexpressing IbCAD1 directed by CaMV 35S promoter were developed to determine the physiological function of IbCAD1. IbCAD1-overexpressing transgenic plants exhibited better plant growth and higher biomass compared to wild type (WT), under normal growth conditions. CAD activity was increased in leaves and roots of transgenic plants. Sinapyl alcohol dehydrogenase activity was induced to a high level in roots, which suggests that IbCAD1 may regulate biosynthesis of syringyl-type (S) lignin. Lignin content was increased in stems and roots of transgenic plants; this increase was in S lignin rather than guaiacyl (G) lignin. Overexpression of IbCAD1 in Arabidopsis resulted in enhanced seed germination rates and tolerance to reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂). Taken together, our results show that IbCAD1 controls lignin content by biosynthesizing S units and plays an important role in plant responses to oxidative stress.

     

Overexpression of the Arabidopsis AtEm6 gene enhances salt tolerance in transgenic rice cell lines

The Arabidopsis thaliana late embryogenesis abundant gene AtEm6 is required for normal seed development and for buffering the rate of dehydration during the latter stages of seed maturation. However, its function in salt stress tolerance is not fully understood. In this investigation, cell suspension cultures of three plant species rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and white pine (Pinus strobes L.) were transformed using Agrobacterium tumefaciens strain LBA4404 harboring pBI-AtEm6. Integration of the AtEm6 gene into the genome of rice, cotton, and white pine has been confirmed by polymerase chain reaction, Southern blotting, and northern blotting analyses. Three transgenic cell lines from each of O. sativa, G. hirsutum, and P. strobus were used to analyze salt stress tolerance conferred by the overexpression of the AtEm6 gene. Our results demonstrated that expression of the AtEm6 gene enhanced salt tolerance in transgenic cell lines. A decrease in lipid peroxidation and an increment in antioxidant enzymes ascorbate peroxidase, glutathione reductase and superoxide dismutase activities were observed in the transgenic cell lines, compared to the non- transgenic control. In rice, AtEM6 increased expression of Ca²⁺-dependent protein kinase genes OsCPK6, OsCPK9, OsCPK10, OsCPK19, OsCPK25, and OsCPK26 under treatment of salt. These results suggested that overexpression of the AtEM6 gene in transgenic cell lines improved salt stress tolerance by regulating expression of Ca²⁺-dependent protein kinase genes. Overexpression of the AtEM6 gene could be an alternative choice for engineering plant abiotic stress tolerance.     

Overexpression of heat shock protein gene PfHSP21.4 in Arabidopsis thaliana enhances heat tolerance

Nuclear-encoded chloroplast small heat shock proteins (Cp-sHSPs) play important roles in plant stress tolerance due to their abundance and diversity. Their functions in Primula under heat treatment are poorly characterized. Here, expression analysis showed that the Primula Cp-sHSP gene, PfHSP21.4, was highly induced by heat stress in all vegetative and generative tissues in addition to constitutive expression in certain development stages. PfHSP21.4 was introduced into Arabidopsis, and its function was analysed in transgenic plants. Under heat stress, the PfHSP21.4 transgenic plants showed increased heat tolerance as shown by preservation of hypocotyl elongation, membrane integrity, chlorophyll content and photosystem II activity (Fv/Fm), increased seedling survival and increase in proline content. Alleviation of oxidative damage was associated with increased activity of superoxide dismutase and peroxidase. In addition, the induced expression of HSP101, HSP70, ascorbate peroxidase and Δ1-pyrroline-5-carboxylate synthase under heat stress was more pronounced in transgenic plants than in wild-type plants. These results support the positive role of PfHSP21.4 in response to heat stress in plants.     

MpAsr encodes an intrinsically unstructured protein and enhances osmotic tolerance in transgenic Arabidopsis

Abscisic acid-, stress- and ripening (ASR) -induced proteins are plant-specific proteins whose expression is up-regulated under abiotic stresses or during fruit ripening. In this study, we characterized an ASR protein from plantain to explore its physiological roles under osmotic stress. The expression pattern of MpAsr gene shows that MpAsr gene changed little at the mRNA level, while the MpASR protein accumulates under osmotic treatment. Through bioinformatic-based predictions, circular dichroism spectrometry, and proteolysis and heat-stability assays, we determined that the MpASR protein is an intrinsically unstructured protein in solution. We demonstrated that the hydrophilic MpASR protein could protect l-lactate dehydrogenase (l-LDH) from cold-induced aggregation. Furthermore, heterologous expression of MpAsr in Escherichia coli and Arabidopsis enhanced the tolerance of transformants to osmotic stress. Transgenic 35S::MpAsr Arabidopsis seeds had a higher germination frequency than wild-type seeds under unfavorable conditions. At the physiological level, 35S::MpAsr Arabidopsis showed increased soluble sugars and decreased cell membrane damage under osmotic stress. Thus, our results suggest that the MpASR protein may act as an osmoprotectant and water-retaining molecule to help cell adjustment to water deficit caused by osmotic stress.     

Costs and benefits of cold tolerance in transgenic Arabidopsis thaliana

Cold tolerance in plants is an ecologically important trait that has been under intensive study for basic and applied reasons. Determining the fitness benefits and costs of cold tolerance has previously been difficult because cold tolerance is normally an induced trait that is not expressed in warm environments. The recent creation of transgenic plants constitutively expressing cold tolerance genes enables the investigation of the fitness consequences of cold tolerance in multiple temperature environments. We studied three genes from the CBF (C-repeat/dehydration responsive element binding factor) cold tolerance pathway, CBF1, 2 and 3, in Arabidopsis thaliana to test for benefits and costs of constitutive cold tolerance. We used multiple insertion lines for each transgene and grew the lines in cold and control conditions. Costs of cold tolerance, as determined by fruit number, varied by individual transgene. CBF2 and 3 overexpressers showed costs of cold tolerance, and no fitness benefits, in both environments. CBF1 overexpressing plants showed no fitness cost of cold tolerance in the control environment and showed a marginal fitness benefit in the cold environment. These results suggest that constitutive expression of traits that are normally induced in response to environmental stress will not always lead to costs in the absence of that stress, and that the ecological risks of CBF transgene escape should be assessed prior to their use in commercial agriculture.     

Overexpression of a ItICE1 gene from Isatis tinctoria enhances cold tolerance in rice

ItICE1, a ICE1-like gene, was isolated from a cDNA library from cold-treated woad (Isatis tinctoria L.) tissues. Expression analysis revealed that the ItICE1 gene was expressed constitutively and was predominant in the leaves of woad seedlings and that its mRNA accumulation was altered by salt stress and abscisic acid application, but not by dehydration and cold stresses. The transgenic rice lines overexpressing ItICE1 showed no growth retardation under normal growth conditions as well as enhanced tolerance to cold stress. Physiological assays showed that ItICE1 not only increased the accumulation of free proline and chlorophyll in transgenic rice lines under cold stress, but also reduced malondialdehyde content and electrolyte leakage. The analysis of gene expression in transgenic rice lines indicated that the maize ubiquitin promoter could respond to cold stress and upregulate ItICE1 gene expression level under its control. Under cold stress conditions, transgenic lines had a remarkably increased expression of OsDREB1A, J013078A14, 001-125-G03, 001-023-B08 and J023042N13 compared to wild-type plants (P < 0.05), implying that ItICE1 functions in the CBF/DREB1 cold-response pathway. These results demonstrate that ItICE1 plays an important regulatory role in the improvement of tolerance to cold stress in rice and is potentially useful for improving the cold tolerance of other plants.     

Overexpression of SaRBP1 enhances tolerance of Arabidopsis to salt stress

Abiotic stresses are the major concern in recent years as their effect on world food production is constantly increasing. We have obtained salt tolerant Arabidopsis lines overexpressing SaRBP1 (Suaeda asparagoides RNA binding protein 1) of a Korean halophyte, S. asparagoides. Homozygous T3 Arabidopsis transgenic lines were developed and used for salt stress tolerance studies. The transgenic seedlings displayed tolerance to salt and mannitol compared to the wild type (WT) seedlings. Transgenic lines produced longer primary roots, more fresh weight, and higher number of lateral roots than WT. In planta stress tolerance assay results showed that the survival rates of transgenic plants were significantly higher than WT plants. Transgenic lines showed delayed germination under 200 mM NaCl stress. In addition, the transgenics showed higher water retention ability than WT. Subcellular localization results revealed that SaRBP1 was targeted to the cytoplasm. Northwestern blot analysis results confirmed the RNA binding property of SaRBP1. Quantitative Real-Time Polymerase Chain Reaction results revealed that many stress marker genes were upregulated by SaRBP1 overexpression. Thus, our data demonstrate that SaRBP1 overexpression lines are tolerant to salt stress. Hence, this is the first report for the functional characterization of SaRBP1, a novel RBP gene isolated from S. asparagoides cDNA library.     

Overexpression of Zostera japonica heat shock protein gene ZjHsp70 enhances the thermotolerance of transgenic Arabidopsis

Molecular Biology Reports - Heat shock protein 70s (Hsp70s) are major members of the heat shock protein family and play a variety of roles to protect plants against stress. Plant Hsp70s are a...