The effects of overexpression of formaldehyde dehydrogenase gene from Brevibacillus brevis on the physiological characteristics of tobacco under formaldehyde stress

The faldh gene encodes the Brevibacillus brevis glutathione-dependent formaldehyde dehydrogenase (FALDH), an enzyme involved in formaldehyde metabolism. In the present work, we have investigated the physiological characteristics of transgenic faldh tobacco under formaldehyde stress. Overexpression of B. brevis FALDH confers tobacco tolerance to high HCHO concentrations. The transgenic tobacco lines had the higher biomass, produced the higher content of total proteins and soluble sugars, the lower levels of MDA, protein carbonyl (PC), and H₂O₂ as compared with the wild-type tobacco under HCHO stress. The contents of chlorophyll (Chl), including Chl a, Chl b, and the ratio of Chl a/b, and the content of anthocyanidin in transgenic plants under HCHO stress were also higher than that in wild-type tobacco. These results show that high HCHO tolerance and changes of physiological characteristics related to stress tolerance were due to the overexpressing of FALDH in tobacco.     

Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants

Expansins are cell wall proteins that are grouped into two main families, α-expansins and β-expansins, and they are implicated in the control of cell extension via the disruption of hydrogen bonds between cellulose and matrix glucans. TaEXPA2 is an α-expansin gene identified in wheat. Based on putative cis-regulatory elements in the TaEXPA2 promoter sequence and the expression pattern induced when polyethylene glycol (PEG) is used to mimic water stress, we hypothesized that TaEXPA2 is involved in plant drought tolerance and plant development. Through transient expression of 35S::TaEXPA2-GFP in onion epidermal cells, TaEXPA2 was localized to the cell wall. Constitutive expression of TaEXPA2 in tobacco improved seed production by increasing capsule number, not seed size, without having any effect on plant growth patterns. The transgenic tobacco exhibited a significantly greater tolerance to water-deficiency stress than did wild-type (WT) plants. We found that under drought stress, the transgenic plants maintained a better water status. The accumulated content of osmotic adjustment substances, such as proline, in TaEXPA2 transgenic plants was greater than that in WT plants. Transgenic plants also displayed greater antioxidative competence as indicated by their lower malondialdehyde (MDA) content, relative electrical conductivity, and reactive oxygen species (ROS) accumulation than did WT plants. This result suggests that the transgenic plants suffer less damage from ROS under drought conditions. The activities of some antioxidant enzymes as well as expression levels of several genes encoding key antioxidant enzymes were higher in the transgenic plants than in the WT plants under drought stress. Collectively, our results suggest that ectopic expression of the wheat expansin gene TaEXPA2 improves seed production and drought tolerance in transgenic tobacco plants.     

The Involvement of Wheat F-Box Protein Gene TaFBA1 in the Oxidative Stress Tolerance of Plants

As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT). The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD), were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants’ tolerance to multiple stress conditions.     

Overexpression of Rice CBS Domain Containing Protein Improves Salinity, Oxidative, and Heavy Metal Tolerance in Transgenic Tobacco

We have recently identified and classified a cystathionine ??-synthase domain containing protein family in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.). Based on the microarray and MPSS data, we have suggested their involvement in stress tolerance. In this study, we have characterized a rice protein of unknown function, OsCBSX4. This gene was found to be upregulated under high salinity, heavy metal, and oxidative stresses at seedling stage. Transgenic tobacco plants overexpressing OsCBSX4 exhibited improved tolerance toward salinity, heavy metal, and oxidative stress. This enhanced stress tolerance in transgenic plants could directly be correlated with higher accumulation of OsCBSX4 protein. Transgenic plants could grow and set seeds under continuous presence of 150?mM NaCl. The total seed yield in WT plants was reduced by 80%, while in transgenic plants, it was reduced only by 15?C17%. The transgenic plants accumulated less Na⁺, especially in seeds and maintained higher net photosynthesis rate and Fv/Fm than WT plants under NaCl stress. Transgenic seedlings also accumulated significantly less H₂O₂ as compared to WT under salinity, heavy metal, and oxidative stress. OsCBSX4 overexpressing transgenic plants exhibit higher abiotic stress tolerance than WT plants suggesting its role in abiotic stress tolerance in plants.     

Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+/K+ and antioxidant competence

High salinity is one of the most serious environmental stresses that limit crop growth. Expansins are cell wall proteins that regulate plant development and abiotic stress tolerance by mediating cell wall expansion. We studied the function of a wheat expansin gene, TaEXPA2, in salt stress tolerance by overexpressing it in tobacco. Overexpression of TaEXPA2 enhanced the salt stress tolerance of transgenic tobacco plants as indicated by the presence of higher germination rates, longer root length, more lateral roots, higher survival rates and more green leaves under salt stress than in the wild type (WT). Further, when leaf disks of WT plants were incubated in cell wall protein extracts from the transgenic tobacco plants, their chlorophyll content was higher under salt stress, and this improvement from TaEXPA2 overexpression in transgenic tobacco was inhibited by TaEXPA2 protein antibody. The water status of transgenic tobacco plants was improved, perhaps by the accumulation of osmolytes such as proline and soluble sugar. TaEXPA2‐overexpressing tobacco lines exhibited lower Na⁺ but higher K⁺ accumulation than WT plants. Antioxidant competence increased in the transgenic plants because of the increased activity of antioxidant enzymes. TaEXPA2 protein abundance in wheat was induced by NaCl, and ABA signaling was involved. Gene expression regulation was involved in the enhanced salt stress tolerance of the TaEXPA2 transgenic plants. Our results suggest that TaEXPA2 overexpression confers salt stress tolerance on the transgenic plants, and this is associated with improved water status, Na⁺/K⁺ homeostasis, and antioxidant competence. ABA signaling participates in TaEXPA2‐regulated salt stress tolerance.     

Over-expression of tobacco <Emphasis Type="Italic">UBC1</Emphasis> encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>)

Ubiquitin (Ub)-conjugating enzyme (UBC, E2) receives Ub from Ub-activating enzyme (E1) and transfers it to target proteins, thereby playing a key role in Ub/26S proteasome-dependent proteolysis. UBC has been reported to be involved in tolerating abiotic stress in plants, including drought, salt, osmotic and water stresses. To isolate the genes involved in Cd tolerance, we transformed WT (wild-type) yeast Y800 with a tobacco cDNA expression library and isolated a tobacco cDNA, NtUBC1 (Ub-conjugating enzyme), that enhances cadmium tolerance. When NtUBC1 was over-expressed in tobacco, cadmium tolerance was enhanced, but the Cd level was decreased. Interestingly, 20S proteasome activity was increased and ubiquitinated protein levels were diminished in response to cadmium in NtUBC1 tobacco. By contrast, proteasome activity was decreased and ubiquitinated protein levels were slightly enhanced by Cd treatment in control tobacco, which is sensitive to Cd. Moreover, the oxidative stress level was induced to a lesser extent by Cd in NtUBC1 tobacco compared with control plants, which is ascribed to the higher activity of antioxidant enzymes in NtUBC1 tobacco. In addition, NtUBC1 tobacco displayed a reduced accumulation of Cd compared with the control, likely due to the higher expression of CAX3 (Ca²⁺/H⁺ exchanger) and the lower expression of IRT1 (iron-responsive transporter 1) and HMA-A and -B (heavy metal ATPase). In contrast, atubc1 and atubc1atubc2 Arabidopsis exhibited lower Cd tolerance and proteasome activity than WT. In conclusion, NtUBC1 expression promotes cadmium tolerance likely by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or the Ub-independent 20S proteasome and by diminishing oxidative stress through the activation of antioxidant enzymes and decreasing Cd accumulation due to higher CAX3 and lower IRT1 and HMA-A/B expression in response to 50 µM Cd challenge for 3 weeks.     

Overexpression of a Triticum aestivum Calreticulin gene (TaCRT1) Improves Salinity Tolerance in Tobacco

Calreticulin (CRT) is a highly conserved and abundant multifunctional protein that is encoded by a small gene family and is often associated with abiotic/biotic stress responses in plants. However, the roles played by this protein in salt stress responses in wheat (Triticum aestivum) remain obscure. In this study, three TaCRT genes were identified in wheat and named TaCRT1, TaCRT2 and TaCRT3-1 based on their sequence characteristics and their high homology to other known CRT genes. Quantitative real-time PCR expression data revealed that these three genes exhibit different expression patterns in different tissues and are strongly induced under salt stress in wheat. The calcium-binding properties of the purified recombinant TaCRT1 protein were determined using a PIPES/Arsenazo III analysis. TaCRT1 gene overexpression in Nicotiana tabacum decreased salt stress damage in transgenic tobacco plants. Physiological measurements indicated that transgenic tobacco plants showed higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) than non-transgenic tobacco under normal growth conditions. Interestingly, overexpression of the entire TaCRT1 gene or of partial TaCRT1 segments resulted in significantly higher tolerance to salt stress in transgenic plants compared with their WT counterparts, thus revealing the essential role of the C-domain of TaCRT1 in countering salt stress in 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.     

Marker-free transgenic durum wheat cv. Karim expressing the AlSAP gene exhibits a high level of tolerance to salinity and dehydration stresses

We have recently isolated the AlSAP (stress-associated protein) gene from the halophyte grass Aeluropus littoralis and demonstrated that AlSAP expression improves tolerance to continuous salt and drought stresses in transgenic tobacco. To extend these findings to an important crop, we generated marker-free transgenic durum wheat plants of the commercial cv. Karim expressing the AlSAP gene. The integration and expression of AlSAP in T3 homozygous plants were ascertained by Southern, Northern and Western blotting respectively. AlSAP wheat lines exhibited improved germination rates and biomass production under severe salinity and osmotic stress conditions. Following a long-term salt or drought stress greenhouse trial, AlSAP lines produced normally filled grains whereas wild-type (WT) plants either died at the vegetative stage under salt stress or showed markedly reduced grain filling under drought stress. Measurements of the RWC (relative water content) and endogenous Na⁺ and K⁺ levels in leaves of AlSAP plants, showed a lower water loss rate and a higher Na⁺ accumulation in senescent-basal leaves, respectively, compared to those of WT plants. Taken together, these results extend to cereals the high potential of the AlSAP gene for engineering effective drought and salt tolerance.     

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.     

Functional analysis of PsG6PDH, a cytosolic glucose-6-phosphate dehydrogenase gene from Populus suaveolens, and its contribution to cold tolerance improvement in tobacco plants

A 1,697-bp cDNA sequence, designated as PsG6PDH, was amplified from Populus suaveolens. Multiple sequence alignment and phylogenetic analysis indicated that PsG6PDH encodes a cytosolic G6PDH isoform, with Southern blot analysis demonstrating that the gene is single or low copy in Populus. Transgenic tobacco plants over-expressing PsG6PDH exhibited enhanced cold tolerance. In both transgenic and wild-type (WT) tobacco plants, cold stress increased leaf malondialdehyde (MDA) content, electrolyte leakage (EL), and peroxide (POD) and superoxide dismutase (SOD) activities; relative to WT, however, transgenic lines had lower MDA content and EL and higher SOD and POD activities. In addition, PsG6PDH activated the expression of stress-related genes, including NtERD10b, NtERD10c, and NtSOD, in tobacco plants. Our results provide evidence regarding PsG6PDH regulatory function in plants during low temperature stress.