Combinational transformation of three wheat genes encoding fructan biosynthesis enzymes confers increased fructan content and tolerance to abiotic stresses in tobacco

Key message

Seven kinds of transgenic tobacco plants transformed with combinations of three FBE genes were obtained. The transgenic plants transformed with Ta1-SST?+?Ta6-SFT genes appeared to have the highest fructan or soluble sugar content and the strongest salt tolerance.

Abstract

Fructan is thought to be one of the important regulators involved in plant tolerance to various abiotic stresses. In this study, wheat-derived genes, Ta1-SST, Ta6-SFT, and Ta1-FFT, encoding fructan biosynthesis enzymes (FBE) were isolated and cloned into vectors modified pBI121 or pZP211. Seven different combinations of the three target genes were transformed into tobacco plants through an Agrobacterium-mediated approach, and transgenic tobacco plants were identified by PCR, ELISA, and Southern blotting. Compared with tobacco plants transformed with other six combinations of the three target genes and with wild-type plants, the transgenic plants transformed with Ta1-SST?+?Ta6-SFT genes contained the highest fructan and soluble sugar content. All seven types of transgenic tobacco plants displayed a much higher level of tolerance to drought, low temperature, and high salinity compared with the wild type. Differences of drought and low temperature tolerance between the transgenic plants containing a single FBE gene and those harboring two or three FBE genes were not significant, but the salt tolerance level of the transgenic plants with different FBE gene combinations from high to low was: Ta1-SST?+?Ta6-SFT?>?Ta1-SST?+?Ta6-SFT?+?Ta1-FFT?>?Ta1-SST?+?Ta1-FFT?>?Ta1-SFT?+?Ta1-FFT?>?single FBE gene. These results indicated that the tolerances of the transgenic tobacco plants to various abiotic stresses were associated with the transformed target gene combinations and the contents of fructan and soluble sugar contained in the transgenic plants.     

Overexpression of Camellia sinensis H1 histone gene confers abiotic stress tolerance in transgenic tobacco

Key message

Overexpression of CsHis in tobacco promoted chromatin condensation, but did not affect the phenotype. It also conferred tolerance to low-temperature, high-salinity, ABA, drought and oxidative stress in transgenic tobacco.

Abstract

H1 histone, as a major structural protein of higher-order chromatin, is associated with stress responses in plants. Here, we describe the functions of the Camellia sinensis H1 Histone gene (CsHis) to illustrate its roles in plant responses to stresses. Subcellular localization and prokaryotic expression assays showed that the CsHis protein is localized in the nucleus, and its molecular size is approximately 22.5 kD. The expression levels of CsHis in C. sinensis leaves under various conditions were investigated by qRT-PCR, and the results indicated that CsHis was strongly induced by various abiotic stresses such as low-temperature, high-salinity, ABA, drought and oxidative stress. Overexpression of CsHis in tobacco (Nicotiana tabacum) promoted chromatin condensation, while there were almost no changes in the growth and development of transgenic tobacco plants. Phylogenetic analysis showed that CsHis belongs to the H1C and H1D variants of H1 histones, which are stress-induced variants and not the key variants required for growth and development. Stress tolerance analysis indicated that the transgenic tobacco plants exhibited higher tolerance than the WT plants upon exposure to various abiotic stresses; the transgenic plants displayed reduced wilting and senescence and exhibited greater net photosynthetic rate (Pn), stomatal conductance (Gs) and maximal photochemical efficiency (Fv/Fm) values. All the above results suggest that CsHis is a stress-induced gene and that its overexpression improves the tolerance to various abiotic stresses in the transgenic tobacco plants, possibly through the maintenance of photosynthetic efficiency.     

The garlic NF-YC gene, <Emphasis Type="Italic">AsNF-YC8</Emphasis>, positively regulates non-ionic hyperosmotic stress tolerance in tobacco

To investigate the relationship between nuclear factor Y (NF-Y) and stress tolerance in garlic, we cloned a NF-Y family gene AsNF-YC8 from garlic, which was largely upregulated at dehydrate stage. Expression pattern analyses in garlic revealed that AsNF-YC8 is induced through abscisic acid (ABA) and abiotic stresses, such as NaCl and PEG. Compared with wild-type plants, the overexpressing-AsNF-YC8 transgenic tobacco plants showed higher seed germination rates, longer root length and better plant growth under salt and drought stresses. Under drought stress, the transgenic plants maintained higher relative water content (RWC), net photosynthesis, lower levels of malondialdehyde (MDA), and less ion leakage (IL) than wild-type control plants. These results indicate the high tolerance of the transgenic plants to drought stress compared to the WT. The transgenic tobacco lines accumulated less reactive oxygen species (ROS) and exhibited higher antioxidative enzyme activities compared with wild-type (WT) plants under drought stress, which suggested that the overexpression of AsNF-YC8 improves the antioxidant defense system by regulating the activities of these antioxidant enzymes, which in turn protect transgenic lines against drought stress. These results suggest that AsNF-YC8 plays an important role in tolerance to drought and salt stresses.     

Increased gibberellin contents contribute to accelerated growth and development of transgenic tobacco overexpressing a wheat ubiquitin gene

Key message

Overexpressing TaUb2 promoted stem growth and resulted in early flowering in transgenic tobacco plants. Ubiquitin are involved in the production, metabolism and proper function of gibberellin.

Abstract

The ubiquitin–26S proteasome system (UPS), in which ubiquitin (Ub) functions as a marker, is a post-translational regulatory system that plays a prominent role in various biological processes. To investigate the impact of different Ub levels on plant growth and development, transgenic tobacco (Nicotiana tabacum L.) plants were engineered to express an Ub gene (TaUb2) from wheat (Triticum aestivum L.) under the control of cauliflower mosaic virus 35S promoter. Transgenic tobacco plants overexpressing TaUb2 demonstrated an accelerated growth rate at early stage and an early flowering phenotype in development. The preceding expression of MADS-box genes also corresponded to the accelerated developmental phenotypes of the transgenic tobacco plants compared to that of wild-type (WT). Total gibberellin (GA) and active GA contents in transgenic tobacco plants were higher than those in WT at the corresponding developmental stages, and some GA metabolism genes were upregulated. Treatment with GA₃ conferred a similarly accelerated grown rate in WT plants to that of transgenic tobacco plants, while growth was inhibited when transgenic tobacco plants were treated with a GA biosynthesis inhibitor. Thus, the results suggest that Ub are involved in the production, metabolism and proper function of GA, which is important in the regulation of plant growth and development.     

Overexpression of tomato GDP-l-galactose phosphorylase gene in tobacco improves tolerance to chilling stress

Key message

The overexpression of tomato GDP- l -galactose phosphorylase gene enhanced tolerance to chilling stress and reduced photoinhibition of photosystems I and II in transgenic tobacco.

Abstract

Chilling stress is a crucial factor that limits the geographical distribution and yield of chilling-sensitive plants. Ascorbate (AsA) protects plants by scavenging reactive oxygen species and reduces photoinhibition by promoting the conversion of violaxanthin to zeaxanthin in the xanthophyll cycle to dissipate excess excitation energy. Possible mechanisms of AsA for plant photoprotection under chilling stress were investigated by isolating the tomato GDP-l-galactose phosphorylase gene (SlGGP) and producing transgenic tobacco plants with overexpression of SlGGP. The transgenic plants subjected to chilling stress accumulated less H₂O₂, demonstrated lower levels of ion leakage and malondialdehyde, and acquired higher net photosynthetic rate, higher maximum photochemical efficiency of PSII, and higher D1 protein content compared with the wild-type (WT) plants. The transgenic plants subjected to chilling stress also showed higher GDP-l-galactose phosphorylase activity, increased AsA content as well as ascorbate peroxidase and oxidizable P700 activities than WT plants. Thus, SlGGP overexpression is crucial in promoting AsA synthesis and alleviating photoinhibition of two photosystems.     

Effect of localized reduction of gibberellins in different tobacco organs on drought stress tolerance and recovery

Drought resistance is increased in plants by the absence of the hormone gibberellic acid (GA) or by a lack of GA sensitivity. We studied the effects of tissue-specific reduction in GA levels on drought tolerance, on recovery from drought stress, and on primary and secondary growth using transgenic tobacco plants expressing the GA-inactivating gene PtGA2ox ₁ (GA 2-oxidase) specifically in leaves, stems, or roots. Localized reduction of bioactive GA₁ levels was achieved by tissue-specific expression of the PtGA2ox ₁ gene in leaves using the rbcs promoter (LD plants), in roots using the TobRB7 promoter (RD plants), and in stems using the LMX5 promoter (SD plants). In response to drought stress, all transgenic tobacco plants exhibited reduced primary and secondary growth and increased drought tolerance with a corresponding reduction in malondialdehyde levels, higher relative water content, increased proline and sugar content, and elevated peroxidase, superoxide dismutase, and catalase activities relative to wild-type plants. The highest level of drought tolerance and the most rapid recovery from stress was achieved by localized reduction of GA₁ in the roots of the RD transgenic plants. In addition, although the total bioactive GA₁ content in RD and LD plants was essentially identical, the heights of LD plants were significantly greater and drought tolerance was significantly less than in RD plants. It is possible that the site of gibberellin-related gene expression plays an important role in the balance between growth and drought tolerance.     

Enhanced salt tolerance of transgenic tobacco plants by co-expression of PcINO1 and McIMT1 is accompanied by increased level of myo-inositol and methylated inositol

Introgression and functional expression of either the PcINO1 (l-myo-inositol 1-phosphate synthase or MIPS coding gene from the wild halophytic rice, Porteresia coarctata) or McIMTI (inositol methyl transferase, IMTI coding gene from common ice plant Mesembryanthemum crystallinum) has earlier been shown to confer salt tolerance to transgenic tobacco plants (Sheveleva et al., Plant Physiol 115:1211–1219, 1997; Majee et al., J Biol Chem 279:28539–28552, 2004). In this communication, we show that transgenic tobacco plants co-expressing PcINO1 and McIMT1 gene either in cytosol or in chloroplasts accumulate higher amount of total inositol (free and methyl inositol) compared to non-transgenic plants. These transgenic plants were more competent in terms of growth potential and photosynthetic activity and were less prone to oxidative stress under salt stress. A positive correlation between the elevated level of total inositol and methylated inositol and the capability of the double transgenic plants to withstand a higher degree of salt stress compared to the plants expressing either PcINO1 or McIMT1 alone is inferred.     

Production of dammarenediol-II triterpene in a cell suspension culture of transgenic tobacco

Key message

Dammarenediol-II is biologically active tetracyclic triterpenoid, which is basic compound of ginsenoside saponin. Here, we established the dammarenediol-II production via a cell suspension culture of transgenic tobacco overexpressing PgDDS.

Abstract

Dammarenediol-II synthase catalyzes the cyclization of 2,3-oxidosqualene to dammarenediol-II, which is the basic triterpene skeleton in dammarene-type saponin (ginsenosides) in Panax ginseng. Dammarenediol-II is a useful candidate both for pharmacologically active triterpenes and as a defense compound in plants. Dammarenediol-II is present in the roots of P. ginseng in trace amounts because it is an intermediate product in triterpene biosynthesis. In this work, we established the production of dammarenediol-II via cell suspension culture of transgenic tobacco. The dammarenediol-II synthase gene (PgDDS) isolated from P. ginseng was introduced into the Nicotiana tobacum genome under the control of 35S promoter by Agrobacterium-mediated transformation. Accumulation of dammarenediol-II in transgenic tobacco plants occurred in an organ-specific manner (roots > stems > leaves > flower buds), and transgenic line 14 (T14) exhibited a high amount (157.8 μg g^?1 DW) of dammarenediol-II in the roots. Dammarenediol-II production in transgenic tobacco plants resulted in reduced phytosterol (β-sitosterol, campesterol, and stigmasterol) contents. A cell suspension culture was established as a shake flask culture of a callus derived from root segments of transgenic (T14) plants. The amount of dammarenediol-II production in the cell suspension reached 573 μg g^?1 dry weight after 3 weeks of culture, which is equivalent to a culture volume of 5.2 mg dammarenediol-II per liter. Conclusively, the production of dammarenediol-II in a cell suspension culture of transgenic tobacco can be applied to the large-scale production of this compound and utilized as a source of pharmacologically active medicinal materials.     

Lysine racemase: a novel non-antibiotic selectable marker for plant transformation

A non-antibiotic based selection system using l-lysine as selection agent and the lysine racemase (lyr) as selectable marker gene for plant transformation was established in this study. l-lysine was toxic to plants, and converted by Lyr into d-lysine which would subsequently be used by the transgenic plants as nitrogen source. Transgenic tobacco and Arabidopsis plants were successfully recovered on l-lysine medium at efficiencies of 23 and 2.4%, respectively. Phenotypic characterization of transgenic plants clearly revealed the expression of normal growth and developmental characteristics as that of wild-type plants, suggesting no pleiotropic effects associated with the lyr gene. The specific activity of Lyr in transgenic tobacco plants selected on l-lysine ranged from 0.77 to 1.06 mU/mg protein, whereas no activity was virtually detectable in the wild-type plants. In addition, the composition of the free amino acids, except aspartic acid, was not affected by the expression of the lyr gene in the transgenic tobacco plants suggesting very limited interference with endogenous amino acid metabolism. Interestingly, our findings also suggested that the plant aspartate kinases may possess an ability to distinguish the enantiomers of lysine for feedback regulation. To our knowledge, this is the first report to demonstrate that the lysine racemase selectable marker system is novel, less controversial and inexpensive than the traditional selection systems.     

Enhanced tolerance to drought and salt stresses in transgenic faba bean (Vicia faba L.) plants by heterologous expression of the PR10a gene from potato

Key message

We report for the first time that expression of potato PR10a gene in faba bean causes enhanced tolerance to drought and salinity.

Abstract

Grain legumes such as soybean (Glycine max L. Merrill), pea (Pisum sativum L.) and faba bean (Vicia faba L.) are staple sources of protein for human and animal nutrition. Among grain legumes, faba bean is particularly sensitive to abiotic stress (in particular osmotic stress due to lack of water or enhanced soil salinity) and often suffers from severe yield losses. Many stress responsive genes have been reported with an effect on improving stress tolerance in model plants. Pathogenesis-related proteins are expressed by all plants in response to pathogen infection and, in many cases, in response to abiotic stresses as well. The PR10a gene isolated from the potato cultivar Desiree was selected for this study due to its role in enhancing salt and/or drought tolerance in potato, and transferred into faba bean cultivar Tattoo by Agrobacterium tumefaciens-mediated transformation system based upon direct shoot regeneration after transformation of meristematic cells derived from embryo axes. The transgene was under the control of the constitutive mannopine synthase promoter (p-MAS) in a dicistronic binary vector, which also contained luciferase (Luc) gene as scorable marker linked by internal ribosome entry site elements. Fertile transgenic faba bean plants were recovered. Inheritance and expression of the foreign genes were demonstrated by PCR, RT-PCR, Southern blot and monitoring of Luciferase activity. Under drought condition, after withholding water for 3 weeks, the leaves of transgenic plants were still green, while non-transgenic plants (WT) wilted and turned brown. Twenty-four hours after re-watering, the leaves of transgenic plants remained green, while WT plants did not recover. Moreover, the transgenic lines displayed higher tolerance to NaCl stress. Our results suggested that introducing a novel PR10a gene into faba bean could be a promising approach to improve its drought and salt tolerance ability, and that MAS promoter is not only constitutive, but also wound-, auxin/cytokinin- as well as stress-inducible.     

Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens

Background

Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds.

Results

Using the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability). Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts.

Conclusion

The results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market.     

Overexpression of a novel chrysanthemum Cys2/His2-type zinc finger protein gene DgZFP3 confers drought tolerance in tobacco

A drought stress-responsive Cys2/His2-type zinc finger protein gene DgZFP3 was previously isolated (Liu et al., Afr J Biotechnol 11:7781–7788, 2012b) from chrysanthemum. To assess roles of DgZFP3 in plant drought stress responses, we performed gain-of-function experiment. The DgZFP3-overexpression tobacco plants showed significant drought tolerance over the wild type (WT). The transgenic lines exhibited less accumulation of H₂O₂ under drought stress, more accumulation of proline and greater activities of peroxidase (POD) and superoxide dismutase than the WT under both control conditions and drought stress. In addition, there was greater up-regulation of the ROS-related enzyme genes (NtSOD and NtPOD) and stress-related genes (NtLEA5 and NtDREB) in transgenic lines under normal or drought conditons. Thus DgZFP3 probably plays a positive regulatory role in drought stress response and has the potential to be utilized in transgenic breeding to improve drought stress tolerance in plants.     

Salt stress induced soybean <Emphasis Type="Italic">GmIFS1</Emphasis> expression and isoflavone accumulation and salt tolerance in transgenic soybean cotyledon hairy roots and tobacco

Effects of isoflavones on plant salt tolerance were investigated in soybean (Glycine max L. Merr. cultivar N23674) and tobacco (Nicotiana tabacum L.). Leaf area, fresh weight, net photosynthetic rate (Pn), and transpiration rate (Tr) of soybean N23674 plants treated with 80 mM NaCl were significantly reduced, while a gene (GmIFS1) encoding for 2-hydroxyisoflavone synthase was highly induced, and isoflavone contents significantly increased in leaves and seeds. To test the impact of isoflavones to salt tolerance, transgenic soybean cotyledon hairy roots expressing GmIFS1 (hrGmIFS1) were produced. Salt stress slightly increased isoflavone content in hairy roots of the transgenic control harboring the empty vector but substantially reduced the maximum root length, root fresh weight, and relative water content (RWC). The isoflavone content in hrGmIFS1 roots, however, was significantly higher, and the above-mentioned root growth parameters decreased much less. The GmIFS1 gene was also transformed into tobacco plants; plant height and leaf fresh weight of transgenic GmIFS1 tobacco plants were much greater than control plants after being treated with 85 mM NaCl. Leaf antioxidant capacity of transgenic tobacco was significantly higher than the control plants. Our results suggest that salt stress-induced GmIFS1 expression increased isoflavone accumulation in soybean and improved salt tolerance in transgenic soybean hairy roots and tobacco plants.     

Salicylic acid induces physiological and biochemical changes in Torreya grandis cv. Merrillii seedlings under drought stress

Key message

SA treatment effectively ameliorated the negative effect of moderate drought stress on T. grandis Seedlings through increasing the water content, Pn, proline content, antioxidant enzymes activity and reducing MDA.

Abstract

Water availability is one of the most critical factors that limits the growth and development of plants. Salicylic acid (SA) is an important signal molecule that modulates plant responses to abiotic stress. To elucidate the regulating mechanism of exogenous SA on Torreya grandis cv. Merrillii under different water stresses, a pot experiment was conducted in a greenhouse. Exposure of T. grandis seedlings to drought conditions resulted in reduced growth rate that was associated with a decline in water content and CO₂ assimilation. Foliar application of SA effectively increased the water content, net CO₂ assimilation rate, proline content and antioxidant enzymes activity in the plants, which helped T. grandis to acclimate to moderate drought stress and increase the shoot dry matter. However, when the plants were under severe drought stress, the relative water content and CO₂ assimilation in the SA-treated plants were significantly lower than those in the control plants. Therefore, our results indicated that SA can effectively ameliorate the negative effect of moderate drought stress on T. grandis seedling growth.