Stress-inducible SWPA2 Promoter Research Articles

Large-scale production of recombinant miraculin protein in transgenic carrot callus suspension cultures using air-lift bioreactors

Miraculin, derived from the miracle fruit (Synsepalum dulcificum), is a taste-regulating protein that interacts with human sweet-taste receptors and transforms sourness into sweet taste. Since miracle fruit is cultivated in West Africa, mass production of miraculin is limited by regional and seasonal constraints. Here, we investigated mass production of recombinant miraculin in carrot (Daucus carota L.) callus cultures using an air-lift bioreactor. To increase miraculin expression, the oxidative stress-inducible SWPA2 promoter was used to drive the expression of miraculin gene under various stress treatments. An 8 h treatment of hydrogen peroxide (H2O2) and salt (NaCl) increased the expression of miraculin gene by fivefold compared with the untreated control. On the other hand, abscisic acid, salicylic acid, and methyl jasmonate treatments showed no significant impact on miraculin gene expression compared with the control. This shows that since H2O2 and NaCl treatments induce oxidative stress, they activate the SWPA2 promoter and consequently up-regulate miraculin gene expression. Thus, the results of this study provide a foundation for industrial-scale production of recombinant miraculin protein using transgenic carrot cells as a heterologous host.

Increased brazzein expression by abiotic stress and bioreactor culture system for the production of sweet protein, brazzein

Plant-based recombinant protein production systems are powerful tools that can be used instead of microbial and animal cell culture systems. The aim of this study was to establish a stable brazzein production system from carrot cell suspension using bioreactor. Using Agrobacterium-mediated transformation (stress-inducible SWPA2 promoter and brazzein gene), three transgenic lines (TC1, 11, 12) were generated. During the TC12 culture period, cell proliferation increased rapidly up to 15 days, and the maximum cell division rate (log phase) was reached after 6 days of culture. Fresh weight peaked at day 18, 3.8-fold (7.6 g 100 mL−1) higher than on day 1, and gene expression was tenfold higher on day 27 than on day 0. The brazzein-encoding gene was also analyzed after treatment with various stress factors for increase of brazzein gene expression, and abscisic acid (ABA) and hydrogen peroxide (H2O2) proved most effective. In particular, the gene expression was enhanced 2.5-fold with 220 μM H2O2 and 2.8-fold with 50 μM ABA, compared with controls. Transgenic cells were then transferred to various types of air-lift bioreactor, and the column bioreactor achieved a higher biomass (238.9 g L−1) than cone and balloon bioreactors. These results demonstrate an efficient protein production system for brazzein using an air-lift bioreactor that may be suitable for the use in the food industry.

Overexpression of Arabidopsis YUCCA6 enhances environment stress tolerance and inhibits storage root formation in sweetpotato

Auxin is a major plant hormone that regulates many developmental processes in plants. Arabidopsis thaliana YUCCA6 (AtYUCCA6) mediates the tryptophan-dependent auxin biosynthesis pathway in A. thaliana. To study the effect of auxin in sweetpotato [Ipomoea batatas (L.) Lam], we generated transgenic sweetpotato plants expressing AtYUCCA6 under the control of the oxidative stress-inducible SWPA2 promoter (referred to as SY plants). SY plants displayed high-auxin phenotypic traits such as narrow downward-curled leaves, increased height, and increased node numbers. SY plants showed increased tolerance to 5 μM of methyl viologen-mediated oxidative stress by maintaining a lower ion leakage than wild-type (WT) plants. Moreover, SY plants exhibited higher relative water contents and lower malondialdehyde contents than WT plants under drought stress. Young SY plants also showed inhibition of adventitious root growth. SY plants showed significantly lower storage root formation than WT plants. Our results indicate that AtYUCCA6-mediated auxin overproduction is associated with tolerance to oxidative and drought stress. It also suggests that negatively regulates the formation of storage roots in sweetpotato.

Stress-induced expression of the sweetpotato gene IbLEA14 in poplar confers enhanced tolerance to multiple abiotic stresses

Late embryogenesis abundant (LEA) proteins are small, highly hydrophilic proteins that act as protectors of macromolecules and increase abiotic stress tolerance in plants. We previously reported that overexpressing sweetpotato IbLEA14 under the control of the CaMV 35S promoter increased osmotic and salt stress tolerance in transgenic sweetpotato calli. In this study, we generated transgenic poplar plants (Populus alba × P. glandulosa) expressing IbLEA14 under the control of the oxidative stress-inducible SWPA2 promoter (referred to as SL plants). Among the 15 SL plants obtained, three lines (SL2, SL7, and SL12) were established based on IbLEA14 transcript levels, tolerance to salt stress and Southern blot analysis. The SL plants exhibited less damage in response to methyl viologen-mediated oxidative stress than non-transgenic (NT) plants. SL plants also showed enhanced tolerance to drought, salt, and heat stress, which was associated with higher photosystem II efficiency and lower malondialdehyde levels compared with NT plants. Furthermore, SL plants had higher levels of monolignol biosynthesis-related gene transcripts under drought stress compared with NT plants. Finally, SL plants exhibited increased tolerance to heat stress, which is associated with the high thermostability of IbLEA14 protein. SL plants might be useful for reforestation on global marginal lands, including desertification and reclaimed areas.

Stress Inducible Overexpression of Arabidopsis Nucleotide Diphosphate Kinase 2 Gene Confers Enhanced Tolerance to Salt Stress in Tall Fescue Plants

Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) is an upstream signaling molecule that has been shown to induce stress tolerance in plants. In this study, the AtNDPK2 gene, under the control of a stress-inducible SWPA2 promoter, was introduced into the genome of tall fescue (Festuca arundinacea Schreb.) plants. The induction of the transgene expression mediated by methyl viologen (MV) and NaCl treatments were confirmed by RT-PCR and northern blot analysis, respectively. Under salt stress treatment, the transgenic tall fescue plants (SN) exhibited lower level of H2O2 and lipid peroxidation accumulations than the non-transgenic (NT) plants. The transgenic tall fescue plants also showed higher level of NDPK enzyme activity compared to NT plants. The SN plants were survived at 300 mM NaCl treatment, whereas the NT plants were severely affected. These results indicate that stress-inducible overexpression of AtNDPK2 might efficiently confer the salt stress tolerance in tall fescue plants.

Overexpressing Arabidopsis ABF3 increases tolerance to multiple abiotic stresses and reduces leaf size in alfalfa

Arabidopsis ABSCISIC ACID-RESPONSIVE ELEMENT-BINDING FACTOR 3 (ABF3), a bZIP transcription factor, plays an important role in regulating multiple stress responses in plants. Overexpressing AtABF3 increases tolerance to various stresses in several plant species. Alfalfa (Medicago sativa L.), one of the most important perennial forage crops worldwide, has high yields, high nutritional value, and good palatability and is widely distributed in irrigated and semi-arid regions throughout the world. However, drought and salt stress pose major constraints to alfalfa production. In this study, we developed transgenic alfalfa plants (cv. Xinjiang Daye) expressing AtABF3 under the control of the sweetpotato oxidative stress-inducible SWPA2 promoter (referred to as SAF plants) via Agrobacterium tumefaciens-mediated transformation. After drought stress treatment, we selected two transgenic lines with high expression of AtABF3, SAF5 and SAF6, for further characterization. Under normal conditions, SAF plants showed smaller leaf size compared to non-transgenic (NT) plants, while no other morphological changes were observed. Moreover, SAF plants exhibited enhanced drought stress tolerance and better growth under drought stress treatment, which was accompanied by a reduced transpiration rate and lower reactive oxygen species contents. In addition, SAF plants showed an increased tolerance to salt and oxidative stress. Therefore, these transgenic AtABF3 alfalfa plants might be useful for breeding forage crops with enhanced tolerance to environmental stress for use in sustainable agriculture on marginal lands.

Overexpression of CuZnSOD and APX enhance salt stress tolerance in sweet potato

Abiotic stresses cause accumulation of reactive oxygen species (ROS) in plants, CuZnSOD and APX are first line defenses against ROS caused by oxidative stress. In this study, CuZnSOD and APX were transferred into salt sensitive sweet potato (cv. Xushu 55–2) under control of stress inducible SWPA2 promoter and tolerance to salt stress was evaluated. When 100 mM NaCl was used to treat stem cuttings, transgenic plants showed enhanced tolerance compared to wild type (WT) plants. Rooting was significantly retarded in WT plants whereas all transgenic plants had significantly enhanced root growth under salt stress. Integration of SOD gene was confirmed by southern blot analysis, and the copy number ranged from 1 to 3. The expression levels of CuZnSOD and APX in transgenic plants were significantly increased up to 13.3 and 7.8 folds to WT under salinity conditions, respectively. SOD and APX activity and ROS staining showed enzyme activities of transgenic plants were increased under salt stress. These results show that CuZnSOD and APX have important roles in enhancing the salt tolerance of sweet potato.

Expressing the sweet potato orange gene in transgenic potato improves drought tolerance and marketable tuber production

Potato (Solanum tuberosum L.) is generally considered to be sensitive to drought stress. Even short periods of water shortage can result in reduced tuber production and quality. We previously reported that transgenic potato plants expressing the sweet potato orange gene (IbOr) under the control of the stress-inducible SWPA2 promoter (referred to as SOR plants) showed increased tolerance to methyl viologen-mediated oxidative stress and high salinity, along with increased carotenoid contents. In this study, in an effort to improve the productivity and environmental stress tolerance of potato, we subjected transgenic potato plants expressing IbOr to water-deficient conditions in the greenhouse. The SOR plants exhibited increased tolerance to drought stress under greenhouse conditions. IbOr expression was associated with slightly negative phenotypes, including reduced tuber production. Controlling IbOr expression imparted the same degree of drought tolerance while ameliorating these negative phenotypic effects, leading to levels of tuber production similar to or better than those of wild-type plants under drought stress conditions. In particular, under drought stress, drought tolerance and the production of marketable tubers (over 80g) were improved in transgenic plants compared with non-transgenic plants. These results suggest that expressing the IbOr transgene can lead to significant gains in drought tolerance and tuber production in potato, thereby improving these agronomically important traits.

Transgenic poplar expressing codA exhibits enhanced growth and abiotic stress tolerance

Glycine betaine (GB), a compatible solute, effectively stabilizes the structure and function of macromolecules and enhances abiotic stress tolerance in plants. We generated transgenic poplar plants (Populus alba×Populus glandulosa) expressing a bacterial choline oxidase (codA) gene under the control of the oxidative stress-inducible SWPA2 promoter (referred to as SC plants). Among the 13 SC plants generated, three lines (SC4, SC14 and SC21) were established based on codA transcript levels, tolerance to methyl viologen-mediated oxidative stress and Southern blot analysis. Growth was better in SC plants than in non-transgenic (NT) plants, which was related to elevated transcript levels of auxin-response genes. SC plants accumulated higher levels of GB under oxidative stress compared to the NT plants. In addition, SC plants exhibited increased tolerance to drought and salt stress, which was associated with increased efficiency of photosystem II activity. Finally, SC plants maintained lower levels of ion leakage and reactive oxygen species under cold stress compared to the NT plants. These observations suggest that SC plants might be useful for reforestation on global marginal lands, including desertification and reclaimed areas.

체세포배발생에 의한 IbOr 유전자 형질전환 카사바 개발

Cassava (Manihot esculenta Crantz) is a useful root crop for food, animal feed and various industrial materials including biofuel. Despite of its importance as an industrial crop, the genetic engineering approaches to manipulate transgenic plant development in cassava are limited. In this study, to develop new cultivar with high level of carotenoids and enhanced tolerance to environmental stresses, sweetpotato IbOr gene involved in accumulation of carotenoids was introduced into an Indonesian IDB high-yielding cassava cultivar under the control of oxidative stress-inducible SWPA2 promoter through Agrobacterium-mediated transformation of friable embryogenic calli. The 19 transgenic lines were successfully generated on the basis of gDNA-PCR and IbOr transcript levels for further characterization in terms of carotenoid contents and environmental stresses. Therefore, IbOr transgenic cassava plants may be developed for enhanced biomass production with high levels of carotenoids on marginal lands.

Transgenic poplar expressing Arabidopsis YUCCA6 exhibits auxin-overproduction phenotypes and increased tolerance to abiotic stress

YUCCA6, a member of the YUCCA family of flavin monooxygenase-like proteins, is involved in the tryptophan-dependent IAA biosynthesis pathway and responses to environmental cues in Arabidopsis. However, little is known about the role of the YUCCA pathway in auxin biosynthesis in poplar. Here, we generated transgenic poplar (Populus alba × P. glandulosa) expressing the Arabidopsis YUCCA6 gene under the control of the oxidative stress-inducible SWPA2 promoter (referred to as SY plants). Three SY lines (SY7, SY12 and SY20) were selected based on the levels of AtYUCCA6 transcript. SY plants displayed auxin-overproduction morphological phenotypes, such as rapid shoot growth and retarded main root development with increased root hair formation. In addition, SY plants had higher levels of free IAA and early auxin-response gene transcripts. SY plants exhibited tolerance to drought stress, which was associated with reduced levels of reactive oxygen species. Furthermore, SY plants showed delayed hormone- and dark-induced senescence in detached leaves due to higher photosystem II efficiency and less membrane permeability. These results suggest that the conserved IAA biosynthesis pathway mediated by YUCCA family members exists in poplar.

Expression of both CuZnSOD and APX in chloroplasts enhances tolerance to sulfur dioxide in transgenic sweet potato plants

We have previously reported that transgenic sweet potato (Ipomoea batatas) plants overexpressing both CuZn superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) under the control of a stress-inducible SWPA2 promoter in chloroplasts (referred to as SSA plants) showed increased resistance to methyl viologen-mediated oxidative stress and chilling. To investigate whether SSA plants show enhanced tolerance to air pollutants, they were exposed to 500ppb of sulfur dioxide (SO2). SO2 caused visible damage to the leaves of sweet potato, but damage in the leaves of non-transgenic (NT) plants was more severe than in those of SSA plants. The photosynthetic activity (Fv/Fm) of the SSA plants decreased by only 7% on the 5th day after the treatment, whereas that of NT plants severely decreased by 63% after 5days of recovery. Moreover, the chlorophyll content in the oldest leaf of NT plants decreased by 69%, whereas that of SSA plants remained at a high level. APX activity in NT plants increased about three times under an SO2 stress, and in SSA plants about five times compared to the case with no stress conditions. These results suggest that the overexpression of both CuZnSOD and APX in chloroplasts reduces the oxidative stress derived from SO2.

Transgenic alfalfa plants expressing AtNDPK2 exhibit increased growth and tolerance to abiotic stresses

In this study, we generated and evaluated transgenic alfalfa plants (Medicago sativa L. cv. Xinjiang Daye) expressing the Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) gene under the control of the oxidative stress-inducible SWPA2 promoter (referred to as SN plants) to develop plants with enhanced tolerance to various abiotic stresses. We selected two SN plants (SN4 and SN7) according to the expression levels of AtNDPK2 and the enzyme activity of NDPK in response to methyl viologen (MV)-mediated oxidative stress treatment using leaf discs for further characterization. SN plants showed enhanced tolerance to high temperature, NaCl, and drought stress on the whole-plant level. When the plants were subjected to high temperature treatment (42 °C for 24 h), the non-transgenic (NT) plants were severely wilted, whereas the SN plants were not affected because they maintained high relative water and chlorophyll contents. The SN plants also showed significantly higher tolerance to 250 mM NaCl and water stress treatment than the NT plants. In addition, the SN plants exhibited better plant growth through increased expression of auxin-related indole acetic acid (IAA) genes (MsIAA3, MsIAA5, MsIAA6, MsIAA7, and MsIAA16) under normal growth conditions compared to NT plants. The results suggest that induced overexpression of AtNDPK2 in alfalfa will be useful for increasing biomass production under various abiotic stress conditions.

The expression of BrMDHAR gene in chloroplasts and mitochondria enhances tolerance to freezing stress in Arabidopsis thaliana

In chloroplasts and mitochondria, antioxidant mechanisms include the ascorbate-glutathione cycle, and monodehydroascorbate reductase (MDHAR) is important for regeneration of ascorbate (AsA) from monodehydroascorbate (MDHA). To improve detoxification of reactive oxygen species (ROS), we established a construct of the MDHAR gene from Brassica rapa fused to the targeting signal peptides of Pisum sativum glutathione reductase (GR), which was controlled by a stress-inducible SWPA2 promoter, and introduced this expression system into Arabidopsis thaliana. Transgenic (TG) plants overexpressing BrMDHAR targeted to chloroplasts and mitochondria through signal peptides showed an elevated MDHAR activity and an increased ratio of AsA to dehydroascorbate (DHA) when compared to wild-type (WT) plants under a freezing stress. These led to increased photosynthetic parameters, redox homeostasis, and biomass in TG plants when compared to the WT plants. Our results suggest that the overexpression of the BrMDHAR gene targeted to chloroplasts and mitochondria conferred an enhanced tolerance against the freezing stress, and a stress adaptation of dual-targeted BrMDHAR was better than that of single BrMDHAR.

Ectopic expression of Brassica rapa L. MDHAR increased tolerance to freezing stress by enhancing antioxidant systems of host plants

Ascorbate (vitamin C) plays an important role in detoxification of reactive oxygen species (ROS) in most living organisms. Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is crucial to regeneration of the oxidized form of ascorbate (monodehydroascorbate) so that it can be recycled to maintain ROS scavenging ability. The MDHAR gene from Brassica rapa L. was cloned and introduced into Arabidopsis thaliana (L.) Heynh. to test the hypothesis that enhanced ROS scavenging activity of BrMDHAR alleviates freezing stress. BrMDHAR was expressed under the control of either the CaMV 35S promoter or stress inducible SWPA2 promoter. Ectopic expression of BrMDHAR led to the up-regulation of many antioxidant genes, including APX, DHAR, GR, SOD, GPX, and PRX Q, which are involved in ascorbate–glutathione cycle. And, transgenic plants showed improved stress tolerance against freezing with exhibiting higher levels of chlorophyll content and antioxidant molecules such as ascorbate and glutathione as well as alleviated redox status and malondialdehyde contents. These results suggested that ectopic expression of BrMDHAR conferred improved tolerance to freezing stress not only by simply recycling ascorbate, but also by inducing co-regulation of the ascorbate–glutathione cycle, which in turn enhances the antioxidant capacity of the host plants.

Transgenic poplar expressing AtNDPK2 exhibits enhanced biomass in the LMO field

Nucleoside diphosphate kinase 2 (NDPK2) is known to regulate the expression of antioxidant genes and auxin-responsive genes in plants. Previously, it was noted that the overexpression of Arabidopsis NDPK2 (AtNDPK2) under the control of an oxidative stress-inducible SWPA2 promoter in transgenic poplar (Populus alba <TEX>${\times}$</TEX> P. tremular var. glandulosa) plants (referred to as SN plants) enhanced tolerance to oxidative stress and improved growth (Plant Biotechnol J 9: 34-347, 2011). In this study, growth of transgenic poplar was assessed under living modified organism (LMO) field conditions in terms of biomass in the next year. The growth of transgenic poplar plants increased in comparison with non-transgenic plants. The SN3 and SN4 transgenic lines had 1.6 and 1.2 times higher dry weight in stems than non-transgenic plants at 6 months after planting, respectively. Transgenic poplar also exhibited increased transcript levels of auxin-response genes such as IAA1, IAA2, IAA5 and IAA6. These results suggest that enhanced AtNDPK2 expression increases plant biomass in transgenic poplar through the regulation of auxin-response genes.

Overexpression of 2-cysteine peroxiredoxin enhances tolerance to methyl viologen-mediated oxidative stress and high temperature in potato plants

Oxidative stress is one of the major causative factors for injury to plants exposed to environmental stresses. Plants have developed diverse defense mechanisms for scavenging oxidative stress-inducing molecules. The antioxidative enzyme 2-cysteine peroxiredoxin (2-Cys Prx) removes peroxides and protects the photosynthetic membrane from oxidative damage. In this study, transgenic potato (Solanum tuberosum L. cv. Atlantic) expressing At2-Cys Prx under control of the oxidative stress-inducible SWPA2 promoter or enhanced CaMV 35S promoter (referred to as SP and EP plants, respectively) was generated using Agrobacterium-mediated transformation. The transgenic plants were tested for tolerance to stress. Following treatment with 3 μM methyl viologen (MV), leaf discs from SP and EP plants showed approximately 33 and 15% less damage than non-transformed (NT) plants. When 300 μM MV was sprayed onto whole plants, the photosynthetic activity of SP plants decreased by 25%, whereas that of NT plants decreased by 60%. In addition, SP plants showed enhanced tolerance to high temperature at 42 °C. After treatment at high temperature, the photosynthetic activity of SP plants decreased by about 7% compared to plants grown at 25 °C, whereas it declined by 31% in NT plants. These results indicate that transgenic potato can efficiently regulate oxidative stress from various environmental stresses via overexpression of At2-Cys Prx under control of the stress-inducible SWPA2 promoter.

Transgenic poplar expressing Arabidopsis NDPK2 enhances growth as well as oxidative stress tolerance

Nucleoside diphosphate kinase 2 (NDPK2) is known to regulate the expression of antioxidant genes in plants. Previously, we reported that overexpression of Arabidopsis NDPK2 (AtNDPK2) under the control of an oxidative stress-inducible SWPA2 promoter in transgenic potato and sweetpotato plants enhanced tolerance to various abiotic stresses. In this study, transgenic poplar (Populus alba × Poplus glandulosa) expressing the AtNDPK2 gene under the control of a SWPA2 promoter (referred to as SN) was generated to develop plants with enhanced tolerance to oxidative stress. The level of AtNDPK2 expression and NDPK activity in SN plants following methyl viologen (MV) treatment was positively correlated with the plant's tolerance to MV-mediated oxidative stress. We also observed that antioxidant enzyme activities such as ascorbate peroxidase, catalase and peroxidase were increased in MV-treated leaf discs of SN plants. The growth of SN plants was substantially increased under field conditions including increased branch number and stem diameter. SN plants exhibited higher transcript levels of the auxin-response genes IAA2 and IAA5. These results suggest that enhanced AtNDPK2 expression affects oxidative stress tolerance leading to improved plant growth in transgenic poplar.

Studies on Salt Tolerance of Transgenic Sweetpotato Which Harbors Two Genes Expressing CuZn Superoxide Dismutase and Ascorbate Peroxidase with the Stress-inducible SWPA2 Promoter

In this study, some physiological indexes of leaves in transgenic sweetpotato ( Ipomoea batatas L. cv. Yulmi), which harbors two genes CuZn superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) genes, with the stress-inducible SWPA2 promoter were evaluated under different concentrations of NaCl treatment. The results showed that physiological indexes were no remarkable differences without NaCl stress between the transgenic sweetpotato (TS) and the non-transformed sweet potato (NS). The activity of superoxide dismutase (SOD), ascorbate peroxidease (APX), peroxidase (POD) and catalase (CAT) in leaves of TS was always higher than NS under the same NaCl stress, respectively, and when the concentration NaCl with 100 mmol/L, the enzyme activities was the most significant difference especially. On the other hand, the root length of TS was longer than that of NS. And the decline range of chlorophyll and malonaldehyde (MDA) content in leaves of TS was lower than that of NS. All these results indicated that transgenic sweetpotato had the resistance to salt tolerance. Therefore, there would be a great significance in efficiently utilizing saline land and alleviating the energy crisis by developing and planting transgenic sweetpotato plants with salt tolerance.

Simultaneous expression of choline oxidase, superoxide dismutase and ascorbate peroxidase in potato plant chloroplasts provides synergistically enhanced protection against various abiotic stresses

Plants synthesize compatible solutes such as glycinebetaine (GB) in response to abiotic stresses. To evaluate the synergistic and protective effect of GB, transgenic potato plants expressing superoxide dismutase (SOD) and ascorbate peroxidase (APX) targeting to chloroplasts (referred to as SSA plants) were retransformed with a bacterial choline oxidase (codA) gene to synthesize GB in chloroplast in naturally occurring non-accumulator potato plants (including SSA) under the control of the stress-inducible SWPA2 promoter (referred to as SSAC plants). GB accumulation resulted in enhanced protection of these SSAC plants and lower levels of H(2)O(2) compared with SSA and non-transgenic (NT) plants after methyl viologen (MV)-mediated oxidative stress. Additionally, SSAC plants demonstrated synergistically enhanced tolerance to salt and drought stresses at the whole-plant level. GB accumulation in SSAC plants helped to maintain higher activities of SOD, APX and catalase following oxidative, salt and drought stress treatments than is observed in SSA and NT plants. Conclusively, GB accumulation in SSAC plants along with overexpression of antioxidant genes rendered the plants tolerant to multiple environmental stresses in a synergistic fashion.