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- Author or Editor: Yeh-Jin Ahn x
The expression profile and functional properties of DcHsp17.7, a small heat shock protein from carrot (Daucus carota L.), were examined under cold stress. Immunoblot analysis showed that low temperature (2 °C) induced DcHsp17.7 in vegetative tissues. Differential accumulation of the transcript and protein under the cold suggests that expression of DcHsp17.7 might be controlled at the transcriptional and/or translational levels. To examine the functional properties of DcHsp17.7, the gene was expressed in Escherichia coli. When exposed to 2 °C for 10 days, transformed cells expressing DcHsp17.7 showed 115% cell viability, whereas control cells recorded 24%, suggesting that DcHsp17.7 can confer cold tolerance. The amount of soluble protein under the cold was 83% in transformed cells expressing DcHsp17.7, whereas the control cells showed only 52%, suggesting that DcHsp17.7 functions as a molecular chaperone preventing cold-induced protein degradation. Native-polyacrylamide analysis revealed that DcHsp17.7 was found in two oligomeric complexes (≈160 and 240 kDa) and possibly multiple complexes (from 300 to 450 kDa) in cold-stressed carrot and transformed E. coli, respectively. During prolonged cold stress, these complexes disappeared and then reappeared, suggesting that the dissociation and reassociation of DcHsp17.7 complexes might be important for the function of the protein.
A small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the Escherichia coli chromosome by RecE/RecT-based homologous recombination to increase cell viability during industrial fermentation, which frequently encounters adverse growth conditions. DNA construct “lipoprotein (Lpp) gene promoter—Hsp17.7 gene—flippase recombination target (Frt) cassette” flanked by the sequences of the insertion site of the E. coli chromosome (yddE pseudogene) was generated by polymerase chain reaction (PCR). The transformed E. coli cell lines that heterologously expressed Hsp17.7 exhibited shorter lag phase, compared with control cell line under normal (37 °C), heat (45 °C), and antifoam conditions. Cell viability was higher in the transformed cell lines in the heat (50 °C, up to 2-fold) and cold (2 °C, up to 1.7-fold) conditions. The soluble protein levels were also higher in the transformed E. coli cell lines by up to 20%, compared with control cell line in both stress conditions. The stress-tolerant transgenic cell lines developed in this study can contribute to more efficient and cost saving industrial cultivation of E. coli, which is most frequently used for recombinant protein production.
The expression and function of DcHsp17.7, a small heat shock protein (sHSP), in carrot (Daucus carota L.) was examined under lead [Pb(II)] and arsenic (arsenate) stresses. In a time course experiment, the level of DcHsp17.7 increased in carrot leaf tissue treated with lead ions or arsenate. To examine the function of DcHsp17.7, the DcHsp17.7 gene was cloned and introduced into Escherichia coli. Heterologous expression of DcHsp17.7 was confirmed by immunoblot analysis using a polyclonal antibody raised against DcHsp17.7. Lead ion and arsenate reduced bacterial cell viability. However, transgenic E. coli with accumulated DcHsp17.7 showed higher levels of survival under both lead ion and arsenate conditions compared with the vector control. Immunoblot analysis showed that the level of heterologously expressed DcHsp17.7 decreased under lead ion conditions, but remained the same under arsenate conditions. Our results suggest that DcHsp17.7 can confer tolerances to lead and arsenic stresses.
The expression and function of DcHsp17.7, a small heat shock protein expressed in carrot (Daucus carota L.), was examined under oxidative and osmotic stress conditions. Comparative analysis revealed that DcHsp17.7 is a cytosolic Class I protein. Sequence alignment showed that DcHsp17.7 has the characteristic α-crystalline domain-containing consensus regions I and II. Under oxidative [hydrogen peroxide (H2O2)] and osmotic (polyethylene glycol) stress conditions, DcHsp17.7 accumulated in carrot leaf tissue. To examine its function under these abiotic stress conditions, the coding sequence of DcHsp17.7 was introduced into Escherichia coli and expressed by isopropyl β-D-1-thiogalactopyranoside treatment. Under both oxidative and osmotic stress conditions, heterologously expressed DcHsp17.7 enhanced bacterial cell viability. The expression level of soluble proteins was higher in transgenic cells expressing DcHsp17.7 when compared with controls under these stress conditions. These results suggest that DcHsp17.7 confers tolerance to both oxidative and osmotic stresses and thereby functions as a molecular chaperone during the stresses examined.
An efficient plant regeneration protocol using cotyledon explants was established for castor (Ricinus communis L.), an important oilseed crop. Mature seed-derived cotyledon explants produced adventitious shoots when placed on Murashige and Skoog (MS) medium containing thidiazuron (TDZ). The rate of shoot regeneration was maximal (≈25 shoots per explant) when explants were cultured on shoot induction medium supplemented with 5 μm TDZ and preincubated in the dark for the first 7 days before transferring to the day/night cycle (16/8 h). Only the proximal ends of cotyledon explants produced adventitious shoots, although green calli were observed in cotyledon veins. After 4 weeks in culture, explants with well-developed shoot buds were transferred to MS medium without plant growth regulators for the shoot elongation and development. At ≈4 months after culture initiation, shoots (2 cm in length) were transferred to root induction medium (MS medium supplemented with 5 μm indole-3-butyric acid) where they developed roots in 4 to 6 weeks. Plantlets were transferred to soil and acclimatized to greenhouse conditions. Histological analysis showed the adventitious induction of the shoots originated from the cortical and epidermal cell layers of the cotyledon explants.
DcHSP17.7, a small heat shock protein from carrot (Daucus carota L.), was expressed in Escherichia coli to examine its functional mechanism under heat stress. When transformed cells expressing DcHSP17.7 were exposed to 50 °C for 1 h, the number of viable cells was ≈4-fold higher than that of control. When the amount of soluble proteins was compared, it was more than twofold higher in transformed cells expressing DcHSP17.7 than that in control, suggesting that DcHSP17.7 may function as a molecular chaperone preventing heat-inducible protein degradation. Native-PAGE followed by immunoblot analysis showed that in transformed E. coli, DcHSP17.7 was present in an oligomeric complex, ≈300 kDa in molecular mass, on isopropyl b-D-thiogalactopyranoside treatment. However, the complex rapidly disappeared when bacterial cells were exposed to heat stress. In carrot, DcHSP17.7 was found in the similar-sized complex (≈300 kDa), but only during heat stress (40 °C), suggesting that the functional structure of DcHSP17.7 may be different in transformed E. coli from that in carrot.
The expression of a small heat shock protein (sHSP) in plants and its possible function in conditions related to nanomaterial exposure were examined. Multiwalled carbon nanotubes (MWCNTs) and silver nanoparticles (AgNPs) induced toxicity that was indicated by the bending and curling of carrot leaf tissues. Both nanomaterials induced the expression of a small heat shock protein in carrot, DcHsp17.7, but reduced the level of a constitutive heat shock cognate 70. To examine the possible function of DcHsp17.7, the coding gene was heterologously expressed in Escherichia coli. Both nanomaterials reduced the viability of E. coli cell lines. However, the transgenic cell line heterologously expressing DcHsp17.7 showed higher levels of cell viability, compared with vector controls, when exposed to MWCNTs and, more notably, to AgNPs. To the best of our knowledge, this is the first study reporting the influence of nanomaterials on the expression of a plant sHSP and its possible function in conferring tolerance to nanomaterial stress.