Heterologous expression of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> sHSP20 confers temperature stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Small heat shock proteins (sHSPs) are heat shock proteins sized 12–43 kDa that can protect proteins from denaturation, particularly under high temperature; sHSPs thus increase the heat tolerance capability of an organisms enabling survival in adverse climates. sHSP20 is overexpressed in Oenococcus oeni in response to low temperatures. However, we found that overexpression of sHSP20 in Escherichia coli BL21 increased the microbial survival ratio at 50 °C by almost 2 h. Adding sHSP20 to the glutamate dehydrogenase solution significantly increased the stability of the enzyme at high temperature (especially at 60–70 °C), low pH values (especially below 6.0), and high concentration of metal ions of Ga²⁺, Zn²⁺, Mn²⁺, and Fe³⁺. Notably, the coexpression of sHSP20 significantly enhanced soluble expression of laccase from Phomopsis sp. XP-8 (CCTCCM209291) in E. coli without codon optimization, as well as the activity and heat stability of the expressed enzyme. In addition to the chaperone activity of sHSP20 in the gene containing host in vivo and the enzyme heat stability in vitro, our study indicated the capability of coexpression of sHSP20 to increase the efficiency of prokaryotic expression of fungal genes and the activity of expressed enzymes.

Open image in new window

Graphical abstract ?

     

Heterologous expression of <Emphasis Type="Italic">ApGSMT2</Emphasis> and <Emphasis Type="Italic">ApDMT2</Emphasis> genes from <Emphasis Type="Italic">Aphanothece halophytica</Emphasis> enhanced drought tolerance in transgenic tobacco

The glycine-methylation biosynthetic pathway of glycinebetaine (GB) has been investigated, but only a few studies on GB accumulation in transgenic higher plants have utilized this pathway. In this study, two methyltransferase genes named ApGSMT2 and ApDMT2, encoding proteins catalyzing GB biosynthesis from glycine, were cloned from a relative strain of Aphanothece halophytica. The potential roles of ApGSMT2 and ApDMT2 in GB synthesis were first examined in transgenic Escherichia coli, which had increased levels of GB and improved salt tolerance. Then ApGSMT2 and ApDMT2 were transferred into tobacco. Compared with transgenic tobacco expressing betA, transgenic tobacco co-expressing ApGSMT2 and ApDMT2 accumulated more GB and exhibited enhanced drought resistance with better germination performance, higher relative water content, less cell membrane damage and better photosynthetic capacity under drought stress. We concluded that the ApGSMT2 and ApDMT2 genes cloned in this study will be very useful for engineering GB-accumulating transgenic plants with enhanced drought resistance.     

<Emphasis Type="Italic">Piriformospora indica</Emphasis>-mediated salinity tolerance in <Emphasis Type="Italic">Aloe vera</Emphasis> plantlets

Piriformospora indica, a root endophytic fungus, has been reported to promote growth of many plants under normal condition and allow the plants to survive under stress conditions. However, its impact on an important medicinal plant Aloe vera L. has not been well studied. Therefore, this study was undertaken to investigate the effect of P. indica on salinity stress tolerance of A. vera plant. P. indica inoculated and non-inoculated A. vera plantlets were subjected to four levels of salinity treatment- 0, 100, 200 and 300 mM NaCl. The salinity stress decreased the ability of the fungus to colonize roots of A. vera but the interaction of A. vera with P. indica resulted in an overall increase in plant biomass and greater shoot and root length as well as number of shoots and roots. The photosynthetic pigment (Chl a, Chl b and total Chl) and gel content were significantly higher for the fungus inoculated A. vera plantlets, at respective salinity concentrations. Furthermore, the inoculated plantlets had higher phenol, flavonoid, flavonol, aloin contents and radical scavenging activity at all salinity concentrations. The higher phenolic and flavonoid content may help the plants ameliorate oxidative stress resulting from high salinity.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">indica</Emphasis> rice cv. ADT 43

A reproducible and highly efficient protocol for Agrobacterium tumefaciens-mediated transformation of indica rice (Oryza sativa L. subsp. indica cv. ADT 43) was established. Prior to transformation, embryogenic callus were induced from mature seeds incubated on Linsmaier and Skoog (LS) medium supplemented with 2.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ thiamine-HCl. Callus, intact mature seeds, and other in vitro derived explants (leaf bases, leaf blades, coleoptiles, and root-tips) were immersed in a bacterial suspension culture of A. tumefaciens strain EHA 105, OD600 of 0.8, and co-cultivated on LS medium for 2 days in the dark at 25 ± 2°C. Based on GUS expression analysis, 10 min incubation time of explants on a co-cultivation medium containing 100 μM acetosyringone was optimum. Following β-glucuronidase (GUS) assay and polymerase chain reaction (PCR) analysis, transformants were identified. Stable integration of the transgene was confirmed in four putatively transformed T₀ plants by Southern blot analysis. The copy number of the transgene in these lines, one to two, was then determined. Among the observations made, necrosis of co-cultivated explants was a problem, as well as sensitivity of callus to Agrobacterium infection. Levels of necrosis could be minimized following co-cultivation of explants in a medium consisting of 30% LS and containing 10 g l⁻¹ (14), polyvinyl pyrrolidone, 10% coconut water, and 250 mg l⁻¹ timentin (15:1). This latter medium also increased the final transformation efficiency to 15.33%.     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

Heterologous expression of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">NPR1</Emphasis> (<Emphasis Type="Italic">AtNPR1</Emphasis>) enhances oxidative stress tolerance in transgenic tobacco plants

In Arabidopsis, NPR1 (non-expressor of pathogenesis related genes 1, AtNPR1) functions downstream of salicylic acid (SA) and modulates the SA mediated systemic acquired resistance. It is also involved in a cross talk with the jasmonate pathway that is essential for resistance against herbivores and necrotrophic pathogens. Overexpression of AtNPR1 in transgenic plants resulted in enhanced disease resistance. Recently, tobacco transgenic plants expressing AtNPR1 were shown to be tolerant to the early instars of Spodoptera litura (Meur et al., Physiol Plant 133:765–775, 2008). In this communication, we show that the heterologous expression of AtNPR1 in tobacco has also enhanced the oxidative stress tolerance. The transgenic plants exhibited enhanced tolerance to the treatment with methyl viologen. This tolerance was associated with the constitutive upregulation of PR1, PR2 (glucanase), PR5 (thaumatin like protein), ascorbate peroxidase (APX) and Cu²⁺/Zn²⁺ superoxide dismutase (SOD). This is the first demonstration of the novel function of heterologous expression of AtNPR1 in oxidative stress tolerance in transgenic tobacco.     

Overexpression of <Emphasis Type="Italic">MeDREB1D</Emphasis> confers tolerance to both drought and cold stresses in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Cassava (Manihot esculenta) is an important tropical crop with extraordinary tolerance to drought stress but few reports on it. In this study, MeDREB1D was significantly and positively induced by drought stress. Two allelic variants of the gene named MeDREB1D(R-2) and MeDREB1D(Y-3) were identified. Overexpressing MeDREB1D(R-2) and MeDREB1D(Y-3) in Arabidopsis resulted in stronger tolerance to drought and cold stresses. Under drought stress, transgenic plants had more biomass, higher survival rates and less MDA content than wild-type plants. Under cold stress, transgenic plants also had higher survival rates than wild-type plants. To further characterize the molecular function of MeDREB1D, we conducted an RNA-Seq analysis of transgenic and wild-type Arabidopsis plants. The results showed that the Arabidopsis plants overexpressing MeDREB1D led to changes in downstream genes. Several POD genes, which may play a vital role in drought and cold tolerance, were up-regulated in transgenic plants. In brief, these results suggest that MeDREB1D can simultaneously improve plant tolerance to drought and cold stresses.     

Heterologous expression of the <Emphasis Type="Italic">msp2 gene</Emphasis> from <Emphasis Type="Italic">Marasmius scorodonius</Emphasis>

For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca²⁺, and hemin.     

Marker-assisted breeding of a photoperiod-sensitive male sterile <Emphasis Type="Italic">japonica</Emphasis> rice with high cross-compatibility with <Emphasis Type="Italic">indica</Emphasis> rice

The incomplete fertility of japonica × indica rice hybrids has inhibited breeders’ access to the substantial heterotic potential of these hybrids. As hybrid sterility is caused by an allelic interaction at a small number of loci, it is possible to overcome it by simple introgression at the major sterility loci. Here we report the use of marker-assisted backcrossing to transfer into the elite japonica cv. Zhendao88 a photoperiod-sensitive male sterility gene from cv. Lunhui422S (indica) and the yellow leaf gene from line Yellow249 (indica). The microsatellite markers RM276, RM455, RM141 and RM185 were used to tag the fertility genes S5, S8, S7 and S9, respectively. Line 509S is a true-breeding photoperiod-sensitive male sterile plant, which morphologically closely resembles the japonica type. Genotypic analysis showed that the genome of line 509S comprises about 92% japonica DNA. Nevertheless, hybrids between line 509S and japonica varieties suffer from a level of hybrid sterility, although the line is highly cross-compatible with indica types, with the resulting hybrids expressing a significant degree of heterosis. Together, these results suggest that segment substitution on fertility loci based on known information and marker-assisted selection are an effective approach for utilizing the heterosis of rice inter-subspecies.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Molecular mapping of rice blast resistance gene <Emphasis Type="Italic">Pi-1</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) in the elite <Emphasis Type="Italic">indica</Emphasis> variety Samba mahsuri

Samba mahsuri (BPT 5204) is a cultivar of the medium slender grain indica variety of Oryza sativa grown across India for its high yield and quality. However, this cultivar is susceptible to several diseases and pests including rice blast. The analysis of near isogenic lines indicated the presence of a resistance gene, Pi-1(t) in the donor cultivar C101LAC which is highly resistant to the rice blast fungus Magnaporthe grisea (M. grisea). C101LAC was crossed with susceptible indica rice cultivar (BPT 5204) to generate the mapping population. A mendelian segregation ratio of 3:1 for resistant to susceptible F₂ plants using bulk segregation analysis confirmed the presence of a major gene pi-1(t) by simple sequence repeats marker RM224 to the highly virulent blast isolate DRR 001.     

Overexpression of sugarcane gene <Emphasis Type="Italic">SoSnRK2.1</Emphasis> confers drought tolerance in transgenic tobacco

Key message

Overexpression of SoSnRK2.1 improved drought tolerance and growth of tobacco plants.

Abstract

Sucrose non-fermenting1-related protein kinase 2 (SnRK2) is a key enzyme in regulating ABA signal transduction in plants, and it plays a significant role in response to multiple abiotic stresses. In this research, SoSnRK2.1 gene was cloned from sugarcane variety GT21 and characterized under various stresses. The cloned SoSnRK2.1 gene has a complete open reading frame of 1002 bp, encoding a peptide of 333 amino acids. The amino acid sequence of SoSnRK2.1 has high homology with those of Zea mays and Oryza sativa, which belongs to SnRK2 s families. The expression of SoSnRK2.1 under stresses of drought, PEG, and ABA indicated that this gene is involved in stress responses in sugarcane. To investigate the gene function, fusional SoSnRK2.1-GFP-pBI121 under control of CaMV 35S was transformed into tobacco plants. Growth and morphology of transgenic plants demonstrated that overexpression of SoSnRK2.1 enhanced drought tolerance in tobacco. Transgenic tobacco plants had lower levels of ion leakage (IL), and contents of maleic dialdehyde (MDA) and H₂O₂, with higher activities of three antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and chlorophyll and relative water content (RWC) than those in wide type (WT) tobacco. SoSnRK2.1 was stably transmitted to the next generation via sexual reproduction. Though the data presented here are from a heterologous system, it is highly likely that SoSnRK2.1 is involved in the abiotic stress response in sugarcane and may be playing an important role in regulation of its growth.

     

Heterologous expression of <Emphasis Type="Italic">Vitreoscilla</Emphasis> haemoglobin in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>)

The vhb gene encoding Vitreoscilla haemoglobin (VHb) was transferred to barley with the aim of studying the role of oxygen availability in germination and growth. Previous findings indicate that VHb expression improves the efficiency of energy generation during oxygen-limited growth, and germination is known to be an energy demanding growth stage during which the embryos also suffer from oxygen deficiency. When subjected to oxygen deficiency, the roots of vhb-expressing barley plants showed a smaller increase in alcohol dehydrogenase (ADH) activity than those of the control plants. This indicates that VHb plants experienced less severe oxygen deficiency than the control plants, possibly due to the ability of VHb to substitute ADH for recycling NADH and maintaining glycolysis. In contrast to previous findings, we found that constitutive vhb expression did not improve the germination rate of barley kernels in any of the conditions studied. In some cases, vhb expression even slowed down germination slightly. VHb production also appeared to restrict root formation in young seedlings. The adverse effects of VHb on germination and root growth may be related to its ability to scavenge nitric oxide (NO), an important signal molecule in both seed germination and root formation. Because NO has both cytotoxic and stimulating properties, the effect of vhb expression in plants may depend on the level and role of endogenous NO in the conditions studied. VHb production also affected the levels of endogenous barley haemoglobin, which may explain the relatively moderate effects of VHb in this study.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.