Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field

Drought stress has often caused significant decreases in crop production which could be associated with global warming. Enhancing drought tolerance without a grain yield penalty has been a great challenge in crop improvement. Here, we report the Arabidopsis thaliana galactinol synthase 2 gene (AtGolS2) was able to confer drought tolerance and increase grain yield in two different rice (Oryza sativa) genotypes under dry field conditions. The developed transgenic lines expressing AtGolS2 under the control of the constitutive maize ubiquitin promoter (Ubi:AtGolS2) also had higher levels of galactinol than the non‐transgenic control. The increased grain yield of the transgenic rice under drought conditions was related to a higher number of panicles, grain fertility and biomass. Extensive confined field trials using Ubi:AtGolS2 transgenic lines in Curinga, tropical japonica and NERICA4, interspecific hybrid across two different seasons and environments revealed the verified lines have the proven field drought tolerance of the Ubi:AtGolS2 transgenic rice. The amended drought tolerance was associated with higher relative water content of leaves, higher photosynthesis activity, lesser reduction in plant growth and faster recovering ability. Collectively, our results provide strong evidence that AtGolS2 is a useful biotechnological tool to reduce grain yield losses in rice beyond genetic differences under field drought stress.     

Molecular tailoring of farnesylation for plant drought tolerance and yield protection

Protecting crop yield under drought stress is a major challenge for modern agriculture. One biotechnological target for improving plant drought tolerance is the genetic manipulation of the stress response to the hormone abscisic acid (ABA). Previous genetic studies have implicated the involvement of the beta-subunit of Arabidopsis farnesyltransferase (ERA1) in the regulation of ABA sensing and drought tolerance. Here we show that molecular manipulation of protein farnesylation in Arabidopsis, through downregulation of either the alpha- or beta-subunit of farnesyltransferase enhances the plant's response to ABA and drought tolerance. To test the effectiveness of tailoring farnesylation in a crop plant, transgenic Brassica napus carrying an ERA1 antisense construct driven by a drought-inducible rd29A promoter was examined. In comparison with the non-transgenic control, transgenic canola showed enhanced ABA sensitivity, as well as significant reduction in stomatal conductance and water transpiration under drought stress conditions. The antisense downregulation of canola farnesyltransferase for drought tolerance is a conditional and reversible process, which depends on the amount of available water in the soil. Furthermore, transgenic plants were more resistant to water deficit-induced seed abortion during flowering. Results from three consecutive years of field trial studies suggest that with adequate water, transgenic canola plants produced the same amount of seed as the parental control. However, under moderate drought stress conditions at flowering, the seed yields of transgenic canola were significantly higher than the control. Using protein farnesyltransferase as an effective target, these results represent a successful demonstration of engineered drought tolerance and yield protection in a crop plant under laboratory and field conditions.     

High-efficiency transformation and selective tolerance against biotic and abiotic stress in mulberry, <Emphasis Type="Italic">Morus indica</Emphasis> cv. K2, by constitutive and inducible expression of tobacco <Emphasis Type="Italic">osmotin</Emphasis>

Osmotin and osmotin-like proteins are stress proteins belonging to the plant PR-5 group of proteins induced in several plant species in response to various types of biotic and abiotic stresses. We report here the overexpression of tobacco osmotin in transgenic mulberry plants under the control of a constitutive promoter (CaMV 35S) as well as a stress-inducible rd29A promoter. Southern analysis of the transgenic plants revealed the stable integration of the introduced genes in the transformants. Real-time PCR analysis provided evidence for the expression of osmotin in the transgenic plants under both the constitutive and stress-inducible promoters. Transgenic plants with the stress-inducible promoter were observed to better tolerate salt and drought stress than those with the constitutive promoter. Transgenic plants when subjected to simulated salinity and drought stress conditions showed better cellular membrane stability (CMS) and photosynthetic yield than non-transgenic plants under conditions of both salinity and drought stress. Proline levels were very high in transgenic plants with the constitutive promoter relative to those with the stress-inducible promoter. Fungal challenge undertaken with three fungal species known to cause serious losses to mulberry cultivation, namely, Fusarium pallidoroseum, Colletotrichum gloeosporioides and Colletotrichum dematium, revealed that transgenic plants with osmotin under control of the constitutive promoter had a better resistance than those with osmotin under the control of the stress-inducible promoter. Evaluation in next generation was undertaken by studying bud break in transgenic and non-transgenic plants under simulated drought (2% polyethylene glycol) and salt stress (200 mM NaCl) conditions. The axillary buds of the selected transgenic lines had a better bud break percentage under stressed conditions than buds from non-transgenic mulberry lines. A biotic assay with Bombyx mori indicated that osmotin protein had no undesirable effect on silkworm rearing and feeding. We therefore conclude that 35S transgenic plants are better suited for both abiotic stress also biotic challenges (fungal), while the rd29A transgenic plants are more responsive to drought.     

Expression of cold and drought regulatory protein (CcCDR) of pigeonpea imparts enhanced tolerance to major abiotic stresses in transgenic rice plants

Main conclusion

Transgenic rice expressing pigeonpea Cc CDR conferred high-level tolerance to different abiotic stresses. The multiple stress tolerance observed in CcCDR -transgenic lines is attributed to the modulation of ABA-dependent and-independent signalling-pathway genes.

Stable transgenic plants expressing Cajanus cajan cold and drought regulatory protein encoding gene (CcCDR), under the control of CaMV35S and rd29A promoters, have been generated in indica rice. Different transgenic lines of CcCDR, when subjected to drought, salt, and cold stresses, exhibited higher seed germination, seedling survival rates, shoot length, root length, and enhanced plant biomass when compared with the untransformed control plants. Furthermore, transgenic plants disclosed higher leaf chlorophyll content, proline, reducing sugars, SOD, and catalase activities, besides lower levels of MDA. Localization studies revealed that the CcCDR-GFP fusion protein was mainly present in the nucleus of transformed cells of rice. The CcCDR transgenics were found hypersensitive to abscisic acid (ABA) and showed reduced seed germination rates as compared to that of control plants. When the transgenic plants were exposed to drought and salt stresses at vegetative and reproductive stages, they revealed larger panicles and higher number of filled grains compared to the untransformed control plants. Under similar stress conditions, the expression levels of P5CS, bZIP, DREB, OsLEA3, and CIPK genes, involved in ABA-dependent and-independent signal transduction pathways, were found higher in the transgenic plants than the control plants. The overall results amply demonstrate that the transgenic rice expressing CcCDR bestows high-level tolerance to drought, salt, and cold stress conditions. Accordingly, the CcCDR might be deployed as a promising candidate gene for improving the multiple stress tolerance of diverse crop plants.

     

Enhanced quantitative resistance to Leptosphaeria maculans conferred by expression of a novel antimicrobial peptide in canola (Brassica napus L.)

The novel antimicrobial peptide MiAMP1, originally isolated from the seeds of Macadamia integrifolia, was constitutively expressed in transgenic tobacco and canola plants to test its effect on disease resistance. Analysis of plants transformed with 35S-MiAMP1 construct by northern and western blot analyses demonstrated the presence of MiAMP1 mRNA and the mature peptide in the transgenic plants. The MiAMP1 purified from the leaves of transgenic plants was biologically active with the same in vitro antifungal activity as native MiAMP1 purified from the seeds of macadamia. The effect of MiAMP1 expression on the economically important canola pathogen Leptosphaeria maculans (causal agent of blackleg disease) was evaluated in comparison with an untransformed control line and an azygous segregant derived from one of the transgenic lines. Lesion development on the cotyledons of the inoculated canola seedlings was significantly reduced in the T₂ progeny of seven independently transformed transgenic lines. These results suggested that, transgenic canola expressing MiAMP1 may be useful for the management of blackleg disease.     

cyp11A1 Canola plants under short time heat stress conditions

In order to investigate the high temperature tolerance of spring canola plants (Brassica napus L.) constitutively expressing cyp11A1 gene which encodes bovine cytochrome P450_SCC the growth features were analyzed under short time heat stress (42°C) in growth chamber. Earlier it was documented that results of the heat tolerance test positively correlated with improvement of high temperature resistance in field trial. Higher relative water content (by 13%) and superoxide dismutase (SOD) activity, lower electrolyte leakage (up 1.4-fold) and smaller increase in chlorophyll a and carotenoid contents in cyp11A1 canola leaves in comparison with wild-type plants under stress allowed to conclude cyp11A1 plants are more tolerant to high temperature than the control ones. We suppose that SOD activity increase which revealed in our transgenic canola in normal condition plays the defining role in the biochemical alterations in plant metabolism for the thermotolerance improvement. SOD activity increment could be caused by heterologous cytochrome P450_SCC activity which resulted in the superoxide radical formation. Cyp11A1 canola plants might be resistant to the other stress conditions of different origin.     

Ovexpression of a Vacuolar H<Superscript>+</Superscript>-ATPase c Subunit Gene Mediates Physiological Changes Leading to Enhanced Salt Tolerance in Transgenic Tobacco

H⁺-ATPase subunit c (VHA-c) is involved in the adaptation to environmental stresses, including salt, drought, and heavy metals. However, it remains unclear whether VHA-c can induce a physiological response related to stress tolerance. To investigate this possibility, we generated transgenic tobacco lines overexpressing a V-ATPase subunit c (LbVHA-c1) gene from Limonium bicolor (Bunge) Kuntze. Compared with wild-type (WT) tobacco, superoxide dismutase (SOD) and peroxidase (POD) activities in the transgenic plants were significantly enhanced under salt stress conditions. The level of malondialdehyde (MDA) in the transgenic plants was significantly lower than that in WT plants grown under salt stress conditions. Moreover, the transgenic plants displayed obviously better growth than the WT plants under salt stress. These results suggest that LbVHA-c1 may confer stress tolerance through enhancing POD and SOD activities, and by protecting membranes from damage by decreasing lipid peroxidation under salt stress.     

Aminolevulinic acid and nitric oxide regulate oxidative defense and secondary metabolisms in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under drought stress

To minimize the damaging effects of stresses, plant growth regulators (PGRs) are widely used to sustain the plant life under stress-prone environments. So, a study was carried out to evaluate the response of two canola (Brassica napus L.) cultivars, Dunkeld and Cyclone, to foliar-applied two potential PGRs, nitric oxide (NO) and 5-aminolevulinic acid, under water deficit conditions. In this study, the levels of NO and ALA used were 0.02 and 0.895 mM, respectively. Plants of both canola cultivars were subjected to control (100% field capacity) and water deficit (60% field capacity). Drought stress significantly decreased growth, chlorophyll pigments, relative water contents (RWC), and soluble proteins, while it increased relative membrane permeability (RMP), proline, glycinebetaine (GB), malondialdehyde (MDA), total phenolics, and activities of catalase (CAT) and peroxidase (POD) enzymes in both cultivars. Foliar application of PGRs improved growth, chlorophyll a, GB, total phenolics, CAT activity, and total soluble proteins, while it decreased RMP, MDA, and POD activity in both canola cultivars. Other physio-biochemical attributes such as chlorophyll b, RWC, hydrogen peroxide (H₂O₂) and proline contents as well as superoxide dismutase (SOD) activity remained unaffected due to application of PGRs. So, the results of the present study suggest that exogenous application of NO and ALA could be useful to enhance the drought tolerance of canola plants by up-regulating the oxidative defense system, osmoprotectant accumulation, and minimizing the lipid peroxidation.     

Increased Tolerance to Mn Deficiency in Transgenic Tobacco Overproducing Superoxide Dismutase

The effect of Mn deficiency on plant growth and activities ofsuperoxide dismutase (SOD) was studied in hydroponically-grownseedlings of transgenic tobacco (Nicotiana tabacum L.) engineeredto overexpress FeSOD in chloroplasts or MnSOD in chloroplastsor mitochondria. In comparison to the non-transgenic parentalline, the activity of MnSOD in the lines overproducing MnSODwas 1.6-fold greater, and the activity of FeSOD in the FeSOD-overproducinglines was 3.2-fold greater, regardless of the Mn treatment (deficientor sufficient). The MnSOD activities decreased due to Mn deficiency,while activities of FeSOD and Cu/ZnSOD remained unaffected 25d after transplanting (DAT). With an increased duration of theMn deficiency stress (45 DAT), FeSOD activity decreased, andthat of MnSOD continued to decrease, while Cu/ZnSOD activitysimultaneously increased. Under Mn sufficiency, non-transgenicparental plants had greater shoot biomass than the transgenics;however, when subjected to Mn deficiency stress, non-transgenicparents suffered a proportionally greater growth reduction thantransgenic lines. Thus, overproduction of MnSOD in chloroplastsmay provide protection from oxidative stress caused by Mn deficiency.Copyright 1999 Annals of Botany Company Manganese deficiency, Nicotiana tabacum, superoxide dismutase (SOD), transgenic tobacco.     

Increase of glycinebetaine synthesis improves drought tolerance in cotton

The tolerance to drought stress of the homozygous transgenic cotton (Gossypium hirsutum L.) plants with enhanced glycinebetaine (GB) accumulation was investigated at three development stages. Among the five transgenic lines investigated, lines 1, 3, 4, and 5 accumulated significantly higher levels of GB than the wild-type (WT) plants either before or after drought stress, and the transgenic plants were more tolerant to drought stress than the wild-type counterparts from young seedlings to flowering plants. Under drought stress conditions, transgenic lines 1, 3, 4, and 5 had higher relative water content, increased photosynthesis, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation than WT plants. The GB levels in transgenic plants were positively correlated with drought tolerance under water stress. The results suggested that GB may not only protect the integrity of the cell membrane from drought stress damage, but also be involved in OA in transgenic cotton plants. Most importantly, the seedcotton yield of transgenic line 4 was significantly greater than that of WT plants after drought stress, which is of great value in cotton production.     

RNAi-mediated disruption of squalene synthase improves drought tolerance and yield in rice

About one-third of the world's rice area is in rain-fed lowlands and most are prone to water shortage. The identification of genes imparting tolerance to drought in the model cereal plant, rice, is an attractive strategy to engineer improved drought tolerance not only rice but other cereals as well. It is demonstrated that RNAi-mediated disruption of a rice farnesyltransferase/squalene synthase (SQS) by maize squalene synthase improves drought tolerance at both the vegetative and reproductive stages. Twenty-day-old seedlings of wild type (Nipponbare) and seven independent events of transgenic RNAi lines showed no difference in morphology. When subjected to water stress for a period of 32 d under growth chamber conditions, transgenic positives showed delayed wilting, conserved more soil water, and improved recovery. When five independent events along with wild-type plants were subjected to drought at the reproductive stage under greenhouse conditions, the transgenic plants lost water more slowly compared with the wild type, through reduced stomatal conductance and the retention of high leaf relative water content (RWC). After 28 d of slow progressive soil drying, transgenic plants recovered better and flowered earlier than wild-type plants. The yield of water-stressed transgenic positive plants ranged from 14-39% higher than wild-type plants. When grown in plates with Yoshida's nutrient solution with 1.2% agar, transgenic positives from three independent events showed increased root length and an enhanced number of lateral roots. The RNAi-mediated inactivation produced reduced stomatal conductance and subsequent drought tolerance.     

Response to mild water stress in transgenic Pssu-ipt tobacco

The response of antioxidant enzymes to cyclic drought was studied in control non-transformed tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) and two types of transgenic Pssu-ipt tobacco (grafted on wild rootstock and poorly rooted progeny of F1 generation) grown under different conditions of irradiation (greenhouse, referred as high light, versus growth chamber, referred as low light). Water stress cycles started with plants at two contrasting developmental stages, i.e., at the stage of vegetative growth (young) and at the onset of flowering (old). Drought reduced the growth of SR1 plants compared with transgenic ones, particularly, when treatment started in earlier stage of plant development. Relative leaf water content was significantly lower (below 70%) in all transgenic grafts and plants compared with the wild type, irrespective of age, drought, and growth conditions. The response of antioxidant enzymes was significantly dependent on plant type and plant age; nevertheless, growth conditions and water stress also affected enzyme activities. Contrary to non-transgenic tobacco, where about half of glutathione reductase activity was found in older plants, both transgenic types exhibited unchanged activities throughout plant development and stress treatment. No differences were found in catalase activity, although the growth in the greenhouse caused a moderate increase in all older plants. In contrast to non-transgenic and Pssu-ipt rooted plants, peroxidase activities (ascorbate, guaiacol, and syringaldazine peroxidase) in older Pssu-ipt grafts were up to four times higher, irrespective of growth and stress, nevertheless, the effect seemed to be age-dependent. Superoxide dismutase (SOD) activity was affected particularly by plant age but also by growth conditions. Unlike in older plants, water stress caused an increase of SOD activities in all younger plants. The differences observed in activities of enzymes of intermediary metabolism (i.e., malic enzyme and glucose-6-phosphate dehydrogenase) revealed that transgenic grafts probably compensated differently for a decrease of ATP and NADPH than control and transgenic rooted plants under stress.     

Expression of multiple resistance genes enhances tolerance to environmental stressors in transgenic poplar (Populus × euramericana 'Guariento')

Commercial and non-commercial plants face a variety of environmental stressors that often cannot be controlled. In this study, transgenic hybrid poplar (Populus × euramericana 'Guariento') harboring five effector genes (vgb, SacB, JERF36, BtCry3A and OC-I) were subjected to drought, salinity, waterlogging and insect stressors in greenhouse or laboratory conditions. Field trials were also conducted to investigate long-term effects of transgenic trees on insects and salt tolerance in the transformants. In greenhouse studies, two transgenic lines D5-20 and D5-21 showed improved growth, as evidenced by greater height and basal diameter increments and total biomass relative to the control plants after drought or salt stress treatments. The improved tolerance to drought and salt was primarily attributed to greater instantaneous water use efficiency (WUEi) in the transgenic trees. The chlorophyll concentrations tended to be higher in the transgenic lines under drought or saline conditions. Transformed trees in drought conditions accumulated more fructan and proline and had increased Fv/Fm ratios (maximum quantum yield of photosystem II) under waterlogging stress. Insect-feeding assays in the laboratory revealed a higher total mortality rate and lower exuviation index of leaf beetle [Plagiodera versicolora (Laicharting)] larvae fed with D5-21 leaves, suggesting enhanced insect resistance in the transgenic poplar. In field trials, the dominance of targeted insects on 2-year-old D5-21 transgenic trees was substantially lower than that of the controls, indicating enhanced resistance to Coleoptera. The average height and DBH (diameter at breast height) of 2.5-year-old transgenic trees growing in naturally saline soil were 3.80% and 4.12% greater than those of the control trees, but these increases were not significant. These results suggested that multiple stress-resistance properties in important crop tree species could be simultaneously improved, although additional research is needed to fully understand the relationships between the altered phenotypes and the function of each transgene in multigene transformants.     

A seed coat outer integument-specific promoter for Brassica napus

In search for seed coat-specific promoters for canola (Brassica napus), transgenic plants carrying a 2,121 bp fragment of Arabidopsis thaliana At4g12960 promoter (AtGILTpro) fused to the uidA reporter gene (GUS) were generated. Out of 7 independent events in transgenic canola plants raised, 2 exhibited GUS activity exclusively in the outer integument of the seed coat. GUS activity in other tissues was also observed in the remaining five transformants. Therefore, the AtGILT promoter can be used as a canola seed coat outer integument-specific promoter after the generation and selection of desired transformants from several transgenic lines.     

Improvement of cold tolerance of the half-high bush Northland blueberry by transformation with the <Emphasis Type="Italic">LEA</Emphasis> gene from <Emphasis Type="Italic">Tamarix androssowii</Emphasis>

Previous studies have shown that the late embryogenesis abundant (LEA) gene of Tamarix androssowii can enhance the drought tolerance of transgenic tobacco. In this study, the cloned LEA gene was transformed into half-high bush Northland blueberry in order to enhance its ability to tolerate cold stress. The cephalosporin antibiotics ceftriaxone, cefotaxime and cefazolin were used to optimize transformation of leaf explants, and kanamycin sulfate was used to select for transgenic shoots. PCR and Southern blot analysis confirmed 8 transformants with LEA gene copy numbers ranging from 1 to 7. The LEA chimeric gene was found to be normally transcribed in 6 transgenic lines by RT-PCR. The 8 transgenic lines were tested for cold tolerance by measuring the activities of peroxidase (POD) and superoxide dismutase (SOD), malondialdehyde (MDA) content and relative electrolyte leakage (REL). Overexpression of the LEA gene enhanced the activity of both POD and SOD under low temperature stress conditions. Lipid peroxidation in the transgenic lines was significantly lower than in non-transgenic plants after cold stress, as determined by MDA content and REL. Thus, our findings indicate that the LEA gene confers increased cold tolerance in the Northland blueberry and implicate the metabolic pathways through which it exerts this effect.     

Overexpression of OsHsp17.0 and OsHsp23.7 enhances drought and salt tolerance in rice

Heat shock proteins (Hsps) play an important role in plant stress tolerance. We previously reported that expression of OsHsp17.0 and OsHsp23.7 could be enhanced by heat shock treatment and/or other abiotic stresses. In this paper, stress tolerance assays of transgenic rice plants overexpressing OsHsp17.0 and OsHsp23.7 have been carried out. Both OsHsp17.0-OE and OsHsp23.7-OE transgenic lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to mannitol and NaCl. Phenotypic analysis showed that transgenic rice lines displayed a higher tolerance to drought and salt stress compared to WT plants. In addition, transgenic rice lines showed significantly lower REC, lower MDA content and higher free proline content than WT under drought and salt stresses. These results suggest that OsHsp17.0 and OsHsp23.7 play an important role in rice acclimation to salt and drought stresses and are useful for engineering drought and salt tolerance rice.     

Drought Induces Oxidative Stress and Enhances the Activities of Antioxidant Enzymes in Growing Rice Seedlings

When rice seedlings grown for 10 and 20 days were subjected to in vitro drought stress of −0.5 and −2.0 MPa for 24 h, an increase in the concentration of superoxide anion (O₂^.−), increased level of lipid peroxidation and a decrease in the concentration of total soluble protein and thiols was observed in stressed seedlings compared to controls. The concentration of H₂O₂ as well as ascorbic acid declined with imposition of drought stress, however glutathione (GSH) concentration declined only under severe drought stress. The activities of total superoxide dismutases (SODs) as well as ascorbate peroxidase (APX) showed consistent increases with increasing levels of drought stress, however catalase activity declined. Mild drought stressed plants had higher guaiacol peroxidase (GPX) and chloroplastic ascorbate peroxidase (c-APX) activity than control grown plants but the activity declined at the higher level of drought stress. The activities of enzymes involved in regeneration of ascorbate i.e. monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) were higher in drought stressed plants compared to controls. Results suggest that drought stress induces oxidative stress in rice plants and that besides SOD, the enzymes of ascorbate-glutathione cycle, which have not been studied in detail earlier under stressful conditions, appear to function as important component of antioxidative defense system under drought stress.