rd29A Promoter Research Articles

Nuclear integration of MYB36 and APX-1 genes impart heat tolerance in wheat.

Elevated temperatures during grain filling stage, exceeding the optimal range by 3-4°C, not only results in a substantial yield reduction in wheat by 10-50% but activates disease and insect infestation. In this research, we introduced heat-tolerant MYB36 and APX-1 gene cassettes into wheat, employing an efficient Agrobacterium mediated transformation protocol, demonstrating higher transformation efficiency. The study encompassed the assembly of MYB36 and APX-1 gene cassettes, and confirmation of gene products in Agrobacterium, followed by the transformation of the MYB36 and APX-1 genes into wheat explants. We were able to select transgenic plant with various combinations. The transgenic plants with APX-1 gene alone produced medium sized grain and spike whereas with both APX-1 and MYB36 genes expressed individually under SPS and rd29a promoter respectively showed good tolerance to heat at 32oC at grain filling/milking stage and produced relatively bold grains. While non-transgenic plants grains were wrinkled with thin spike showing susceptibility to heat. This research contributes to the broader scientific understanding of plant stress responses and the combined effectiveness of MYB36 and APX-1 genes in crop improvement without disturbing normal nutritional values. The gene integration can serve as a valuable tool in breeding programs aimed at developing heat-tolerant wheat varieties. These findings also advance our comprehension of the functions of heat-induced genes and lay the foundation for selecting optimal candidates for in-depth functional studies of heat-responsive MYB36 and APX-1 genes in wheat.

Inducible overexpression of MYB118-like gene improves anthocyanin production in Populus × canescens

Anthocyanins are secondary metabolites which contribute different colors to the leaves. Anthocyanin biosynthesis is regulated by MYB transcription factors, which have been extensively studied and characterized in a diversity of plants. In this study, we identified a novel MYB transcription factor MYB118L, from Populus 'Zhonghua Hongye', which showed an elevated accumulation of anthocyanin than Populus clone ZL-2025. Subcellular localization analyses revealed that MYB118L-GFP fusion protein was specifically located in the nucleus. Transgenic plants overexpressing MYB118L driven by the stress-inducible Rd29A promoter showed a significant increase in anthocyanin production, resulting in a red coloration of the leaves under drought stress conditions. These plants also exhibited higher expression levels of genes involved in anthocyanin biosynthesis compared to the wild type, suggesting that MYB118L positively regulates the expression of these genes. Y1H and dual-luciferase assays confirmed that MYB118L can directly activate the promoters of LAR1 gene. Our findings suggest that MYB118L is an essential transcription factor involved in the regulation of anthocyanin biosynthesis in poplar and could be utilized for genetic engineering of colorful tree species.

Ectopic overexpression of ShCBF1 and SlCBF1 in tomato suggests an alternative view of fruit responses to chilling stress postharvest.

Postharvest chilling injury (PCI) is a physiological disorder that often impairs tomato fruit ripening; this reduces fruit quality and shelf-life, and even accelerates spoilage at low temperatures. The CBF gene family confers cold tolerance in Arabidopsis thaliana, and constitutive overexpression of CBF in tomato increases vegetative chilling tolerance, in part by retarding growth, but, whether CBF increases PCI tolerance in fruit is unknown. We hypothesized that CBF1 overexpression (OE) would be induced in the cold and increase resistance to PCI. We induced high levels of CBF1 in fruit undergoing postharvest chilling by cloning it from S. lycopersicum and S. habrochaites, using the stress-inducible RD29A promoter. Harvested fruit were cold-stored (2.5°C) for up to three weeks, then rewarmed at 20°C for three days. Transgene upregulation was triggered during cold storage from 8.6- to 28.6-fold in SlCBF1-OE, and between 3.1- to 8.3-fold in ShCBF1-OE fruit, but developmental abnormalities in the absence of cold induction were visible. Remarkably, transgenic fruit displayed worsening of PCI symptoms, i.e., failure to ripen after rewarming, comparatively higher susceptibility to decay relative to wild-type (WT) fruit, lower total soluble solids, and the accumulation of volatile compounds responsible for off-odors. These symptoms correlated with CBF1 overexpression levels. Transcriptomic analysis revealed that the ripening and biotic and abiotic stress responses were altered in the cold-stored transgenic fruit. Seedlings grown from 'chilled' and 'non-chilled' WT fruit, in addition to 'non-chilled' transgenic fruit were also exposed to 0°C to test their photosynthetic response to chilling injury. Chilled WT seedlings adjusted their photosynthetic rates to reduce oxidative damage; 'non-chilled' WT seedlings did not. Photosynthetic parameters between transgenic seedlings were similar at 0°C, but SlCBF1-OE showed more severe photoinhibition than ShCBF1-OE, mirroring phenotypic observations. These results suggest that 1) CBF1 overexpression accelerated fruit deterioration in response to cold storage, and 2) Chilling acclimation in fructus can increase chilling tolerance in seedling progeny of WT tomato.

Phosphatidic acid produced by phospholipase Dα1 and Dδ is incorporated into the internal membranes but not involved in the gene expression of RD29A in the abscisic acid signaling network in Arabidopsis thaliana.

Core protein components of the abscisic acid (ABA) signaling network, pyrabactin resistance (PYR), protein phosphatases 2C (PP2C), and SNF1-related protein kinase 2 (SnRK2) are involved in the regulation of stomatal closure and gene expression downstream responses in Arabidopsis thaliana. Phosphatidic acid (PA) produced by the phospholipases Dα1 and Dδ (PLDs) in the plasma membrane has been identified as a necessary molecule in ABA-inducible stomatal closure. On the other hand, the involvement of PA in ABA-inducible gene expression has been suggested but remains a question. In this study, the involvement of PA in the ABA-inducible gene expression was examined in the model plant Arabidopsis thaliana and the canonical RD29A ABA-inducible gene that possesses a single ABA-responsive element (ABRE) in the promoter. The promoter activity and accumulation of the RD29A mRNA during ABA exposure to the plants were analyzed under conditions in which the production of PA by PLDs is abrogated through chemical and genetic modification. Changes in the subcellular localization of PA during the signal transduction were analyzed with confocal microscopy. The results obtained in this study suggest that inhibition of PA production by the PLDs does not affect the promoter activity of RD29A. PA produced by the PLDs and exogenously added PA in the plasma membrane are effectively incorporated into internal membranes to transduce the signal. However, exogenously added PA induces stomatal closure but not RD29A expression. This is because PA produced by the PLDs most likely inhibits the activity of not all but only the selected PP2C family members, the negative regulators of the RD29A promoter. This finding underscores the necessity for experimental verifications to adapt previous knowledge into a signaling network model before its construction.

Transformation of SNAC1 under Stress Inducible Promoter Rd29a Confers High Yield and Stress Tolerance in Rice

Abiotic stress tolerance in plants is often induced by activation of transcription factors. An increased stress tolerance was observed in indica rice cultivar BRRI dhan55 after transforming with the transcription factor SNAC1 (stress responsive NAC1) under stress inducible promoter rd29A that minimized unexpected phenotype and metabolic burden in transgenic lines. Tissue culture independent Agrobacterium-mediated in planta transformation method was used. Molecular analyses confirmed the successful integration of the SNAC1 gene and significantly higher gene expression level in the transgenic lines. The transgenic lines showed 3:1 segregation ratio at T2 generation following the Mendelian law of inheritance. Assays for leaf disk senescence and chlorophyll content at 100 mM and 200 mM salt and survival rates at 200 mM salt and drought conditions at 12 days of water withdrawal and after recovery under water showed significantly increased stress tolerance in the transgenics lines at seedling stage compared to the wild type. Enhanced yield, spikelet fertility, and 100 grain weight were observed at the reproductive stage compared to wild type under both salinity and drought stress conditions. Thus, SNAC1 expression in plants under inducible promoter is a better choice to enhance stress tolerance and yield under salinity and drought conditions. Plant Tissue Cult. & Biotech. 33(2): 115-133, 2023 (December)

Wheat Transformation with ScTPS1-TPS2 Bifunctional Enzyme for Trehalose Biosynthesis Protects Photosynthesis during Drought Stress

Wheat cultivation makes an important contribution to human nutrition. Trehalose synthesis plays a role in the tolerance to drought stress. A bifunctional TPS-TPP enzyme gene from yeast was used to obtain transgenic wheat plants to increase trehalose synthesis. Mature wheat embryos were transformed using pGreen rd29A::TPS1-TPS2 or pGreen 35S::TPS1-TPS2 constructs. The transgene presence in mature leaves of T3 plants was confirmed by sequencing a PCR fragment of the inserted transgene. Transgenic and NT plants were submitted to drought stress for eight days. Transformed wheat lines retained a higher relative water content than NT plants during drought stress, and the Rubisco activity was unaffected. Plants transformed with the 35S construct showed a lower photosynthetic rate and lower fructose 1–6-bisphosphatase (FBPase) activity during drought, suggesting that constitutive trehalose and sucrose synthesis caused a reduced ribulose 1,5-bisphosphate (RuBP) regeneration. Lines transformed with the rd29A promoter showed a higher photosynthetic rate after eight days of drought, as the RuBP regeneration was unaffected. Transgenic wheat plants had higher biomass and grain weight than NT plants after drought. These results suggest that trehalose synthesis improves photosynthesis during stress and induces changes in the activity of some Calvin-cycle enzymes, reflected in plant metabolism and growth.

Breeding of Lotus japonicus that can overcome adverse seasonal environment by coupling flowering time and abiotic stresses

AbstractManipulation of floral transition may allow for a free change in the duration of plant vegetative and reproductive growth, thus rendering them to avoid adverse seasonal environmental conditions. In this report, we investigated the effect of the stress‐inducible promoter RD29A on driving the rice FT ortholog Hd3a expression in Lotus japonicus ‘MG20’ in response to environmental changes. Constitutive overexpression of Hd3a (HOE) in ‘MG20’ hastened its flowering in contrast to non‐transformed control (wild type; WT), which did not flower in winter hampered by long days and low temperatures. RD29A promoter had low activity under non‐stress conditions and was induced by cold, drought and salinity in transgenic ‘MG20’. Transgenic plants of Hd3a driven by the RD29A promoter (RH) could flower under unfavourable weather conditions that prohibited the flowering of WT plants. In addition, RH transgenic plants exhibited thriving clumps of branching, inflorescence, and pods because of the low temperature‐induced expression of Hd3a. These results demonstrate that inducible expression of florigen has the potential to tackle unfavourable weather conditions and boost plant production.

RD29A promoter constitutively drives a rice Hd3a expression to promote early-flowering in Saussurea involucrate Kar. et Kir. ex Maxim

S. involucratae, an endemic and endangered plant, is a valuable and traditional Chinese medicinal herb. In order to control the flowering time of S. involucratae, we used the well-known stress inducible RD29A promoter to drive Hd3a (a FT ortholog from rice) expression in S. involucratae. Unexpectedly, the majority of regenerated buds in RD29A::Hd3a transgenic lines (S-RH) produced flowers in tissue culture stage under normal growth (25 ± 2 °C) condition. Their flowering time was not further influenced by salt treatment. Hd3a in S-RH was strongly expressed in MS media supplemented with or without 50 mM NaCl. RD29A::GUS transgenic experiments further revealed that RD29A constitutively promoted GUS expression in both S. involucrate and halophyte Thellungiella halophile, in contrast to glycophic plants Oryza sativa L. ‘Zhonghua 11’, in which its expression was up-regulated by cold, salinity, and drought stress. The results supported the hypothesis that RD29A promoter activity is inducible in stress-sensitive plants, but constitutive in stress-tolerant ones. Importantly, S-RH plants produced pollen grains and seeds under normal conditions. Additionally, we found that OsLEA3-1::Hd3a and HSP18.2::Hd3a could not promote S. involucrate to flower under either normal conditions or abiotic stresses. Taken together, we demonstrated the potential of RD29A::Hd3a might be served as a feasible approach in breeding S. involucrate under normal condition.

Field evaluation of TaDREB2B-ectopic expression sugarcane (Saccharum spp. hybrid) for drought tolerance.

Sugarcane is one of the most crucial sugar crops globally that supplies the main raw material for sugar and ethanol production, but drought stress causes a severe decline in sugarcane yield worldwide. Enhancing sugarcane drought resistance and reducing yield and quality losses is an ongoing challenge in sugarcane genetic improvement. Here, we introduced a Tripidium arundinaceum dehydration-responsive element-binding transcription factor (TaDREB2B) behind the drought-responsible RD29A promoter into a commercial sugarcane cultivar FN95-1702 and subsequently conducted a series of drought tolerance experiments and investigation of agronomic and quality traits. Physiological analysis indicated that Prd29A: TaDREB2B transgenic sugarcane significantly confers drought tolerance in both the greenhouses and the field by enhancing water retention capacity and reducing membrane damage without compromising growth. These transgenic plants exhibit obvious improvements in yield performance and various physiological traits under the limited-irrigation condition in the field, such as increasing 41.9% yield and 44.4% the number of ratooning sugarcane seedlings. Moreover, Prd29A: TaDREB2B transgenic plants do not penalize major quality traits, including sucrose content, gravity purity, Brix, etc. Collectively, our results demonstrated that the Prd29A-TaDREB2B promoter-transgene combination will be a useful biotechnological tool for the increase of drought tolerance and the minimum of yield losses in sugarcane.

Comprehensive genome-wide analysis of polyamine and ethylene pathway genes in Cleistogenes songorica and CsSAMDC2 function in response to abiotic stress

Polyamine and ethylene pathway genes (PEGs) are widely involved in regulating plant abiotic stresses. However, the PEGs in Cleistogenes songorica have not yet been thoroughly studied. In the present study, 17 polyamine-, 12 ethylene- and 6 S-adenosylmethionine (SAM)- related genes were identified at the genome-wide level in C. songorica . Phylogenetic analysis showed that genes in the ethylene and polyamine pathways and SAM family genes were not clearly separated. Promoter regions of these genes were rich in stress-related cis -regulatory elements. Several PEG and SAM genes coexpressed together and were responsive to abiotic stress treatment. In particular, qRT-PCR analysis showed that the expression of CsSAMDC2 increased under salt, dehydration and ABA treatment conditions. Arabidopsis plants transformed with CsSAMDC2 driven by a stress-inducible RD29A or constitutive promoter 35S exhibited enhanced dehydration tolerance and maintained higher chlorophyll content and photosynthetic capacity. The expression of CsSAMDC2 was generally higher in lines harboring RD29A::CsSAMDC2 compared to 35 S::CsSAMDC2 lines under drought stress treatment. Seed germination of RD29A::CsSAMDC2 or 35 S::CsSAMDC2 transgenic plants under salt stress treatment was superior to their wild-type counterparts, and the leaves of the transgenic plants exhibited a more compact structure. In addition, under salt stress, application of ABA improved lateral root development of RD29A::CsSAMDC2 Arabidopsis plants. The qRT-PCR analysis indicated that the expression of well-known abiotic stress-responsive genes AtPEGs, AtERF1 and AtRD29A, increased in both RD29A::CsSAMDC2 and 35 S::CsSAMDC2 transgenic Arabidopsis after drought, salt stress and salt stress plus ABA treatments. Our results suggest that CsSAMDC2 enhances drought and salt tolerance in Arabidopsis and therefore may contribute to abiotic stress tolerance in forage and food crops. • Genome wide analysis of ethylene and polyamine pathway and SAM family genes in C. songorica. • Evolutionary and functional similarities between ethylene and polyamine pathway genes. • CsSAMDC2 driven by RD29A promoter improved drought and salt tolerance of transgenic Arabidopsis. • Application of ABA on transgenic lines under salt stress further improved the expression of polyamine and ethylene pathway genes.

Stress-Inducible Overexpression of SlDDF2 Gene Improves Tolerance against Multiple Abiotic Stresses in Tomato Plant

Dehydration-responsive element-binding protein 1 (DREB1)/C-repeat binding factor (CBF) family plays a key role in plant tolerance against different abiotic stresses. In this study, an orthologous gene of the DWARF AND DELAYED FLOWERING (DDF) members in Arabidopsis, SlDDF2, was identified in tomato plants. The SlDDF2 gene expression was analyzed, and a clear induction in response to ABA treatment, cold, salinity, and drought stresses was observed. Furthermore, two transgenic lines (SlDDF2-IOE#6 and SlDDF2-IOE#9) with stress-inducible overexpression of SlDDF2 under Rd29a promoter were generated. Under stress conditions, the gene expression of SlDDF2 was significantly higher in both transgenic lines. The growth performance, as well as physiological parameters, were evaluated in wild-type and transgenic plants. The transgenic lines showed growth retardation phenotypes and had higher chlorophyll content under stress conditions in plants. However, the relative decrease in growth performance (plant height, leaf number, and leaf area) in stressed transgenic lines was lower than that in stressed wild-type plants, compared with nonstressed conditions. The reduction in the relative water content and water loss rate was also lower in the transgenic lines. Compared with wild-type plants, transgenic lines showed enhanced tolerance to different abiotic stresses including water deficit, salinity, and cold. In conclusion, stress-inducible expression of SlDDF2 can be a useful tool to improve tolerance against multiple abiotic stresses in tomato plants.

Pyramiding ascorbate-glutathione pathway in Lycopersicum esculentum confers tolerance to drought and salinity stress.

Stacking Glutathione-Ascorbate pathway genes (PgSOD, PgAPX, PgGR, PgDHAR and PgMDHAR) under stress inducible promoter RD29A imparts significant tolerance to drought and salinity stress in Solanum lycopersicum. Although the exposure of plants to different environmental stresses results in overproduction of reactive oxygen species (ROS), many plants have developed some unique systems to alleviate the ROS production and mitigate its deleterious effect. One of the key pathways that gets activated in plants is ascorbate glutathione (AsA-GSH) pathway. To demonstrate the effect of this pathway in tomato, we developed the AsA-GSH overexpression lines by stacking the genes of the AsA-GSH pathway genes isolated from Pennisetum glaucoma (Pg) including PgSOD, PgAPX, PgGR, PgDHAR and PgMDHAR under stress inducible promoter RD29A. The overexpression lines have an improved germination and seedling growth with concomitant elevation in the survival rate. The exposure of transgenic seedlings to varying stress regiments exhibited escalation in the antioxidant enzyme activity and lesser membrane damage as reflected by decreased electrolytic leakage and little accumulation of malondialdehyde and H2O2. Furthermore, the transgenic lines accumulated high levels of osmoprotectants with increase in the relative water content. The increased photosynthetic activity and enhanced gaseous exchange parameters further confirmed the enhanced tolerance of AsA-GSH overexpression lines. We concluded that pyramiding of AsA-GSH pathway genes is an effective strategy for developing stress resistant crops.

AtDREB2A Gene Expression Under Control of the Inducible Promoter and Virus 5’-untranslated Regions Improves Tolerance to Salinity in Nicotiana Tabacum

Transcriptional factor DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A (DREB2A) induces the expression of many genes in dehydration, heat shock, and salinity in Arabidopsis. Deletion of sequence coding the 30 amino acid central region transforms full length (FL) protein DREB2A FL into a more stable and constitutively active form known as DREB2A CA. Here, using agrobacteria, a leaf disc transformation of Nicotiana tabacum v. Samsun NN was carried out by transgenes AtDREB2A-FL and AtDREB2A-CA coding the proteins with His-tag on the С-end. The effects of combinations of constitutive 35S CaMV promoter or inducible rd29A promoter with different viral or artificial 5’-untranslated regions (UTR), 5’TMV, 5’PVY, 5’AMV or 5’ARC1, known as translational enhancers were evaluated on the both transgenes’ expression. Using an antibody to His-tag, recombinant protein synthesis was detected in transgenic plants in normal and heat shock conditions. After comparative analysis, it was shown that the properties of different 5’-UTRs vary greatly and depended on separate conjunction of promoter and transgene. The integration of AtDREB2A CA under control of the rd29A promoter and 5’TMV or 5’AMV in genome effectively improved tolerance of tobacco transgenic plants to 400 mM NaCl and to drought.

Stress-inducible Arabidopsis thaliana RD29A promoter constitutively drives Citrus sinensis APETALA1 and LEAFY expression and precocious flowering in transgenic Citrus spp.

Transgenic 'Duncan' grapefruit (Citrus paradisi Macf.) and 'Valencia' sweet orange (Citrus sinensis [L.] Osbeck) plants ectopically expressing C. sinensis (cv. Washington navel orange) APETALA1 (CsAP1) or LEAFY (CsLFY) genes under control of the Arabidopsis thaliana stress-inducible promoter AtRD29A flowered under non-inductive (warm temperature, well-watered) greenhouse conditions, whereas their wild-type (WT) counterparts did not. The transgenic plants that flowered exhibited no altered morphological features, except the lack of thorns characteristic of juvenile WT plants. The most precocious T0 line, 'Duncan' grapefruit (Dun134-3) expressing the CsAP1 gene, flowered and fruited when it was 4.5years old and the T1 siblings from this line flowered and fruited when they were just over 18months old. In contrast, T1 seedlings from three lines of 'Duncan' grapefruit expressing the CsLFY gene flowered within 3months after germination, but were unable to support fruit development. Transcript levels of corresponding transgenes in leaves were not correlated with earliness of flowering. To further study the activity of AtRD29A, leaves from three 'Carrizo' citrange (C. sinensis×Poncirus trifoliata) rootstock seedlings transformed with the green fluorescent protein (GFP) gene under regulation of the AtRD29A promoter were subjected to drought stress or well-watered conditions. Expression of GFP was not stress-dependent, consistent with the observation of flowering of CsAP1 and CsLFY transgenic plants under non-inductive conditions. Taken together, the results suggest that AtRD29A is constitutively expressed in a citrus background. Despite the loss of control over flowering time, transgenic citrus lines ectopically expressing C. sinensis AP1 or LFY genes under control of the A. thaliana RD29A promoter exhibit precocious flowering, fruit development and viable transgenic seed formation. These transformed lines can be useful tools to reduce the time between generations to accelerate breeding.

Controlled Over-Expression of AtDREB1A Enhances Tolerance against Drought and Salinity in Rice

Engineering transcription factors (TF) hold promise in enhancing abiotic stress tolerance in plants. In this study, one of the popular rice varieties of South India, namely ADT 43, was engineered with a TF AtDREB1A driven by a stress-inducible rd29A promoter. PCR and Southern hybridization were employed to confirm the integration and copy number of the transgene. Transgenic lines (T1) of ADT 43 showed enhanced tolerance to drought and salinity compared to the non-transgenic ADT 43. Transgenic lines were found to maintain higher RWC %, lower leaf temperature, and partially closed stomata, enabling better survival under stress conditions. qRT-PCR analysis revealed the strong induction of AtDREB1A transcripts during drought. Transgenic lines of ADT 43 exhibited increased germination and retention of chlorophyll in their leaves under salinity. Evaluation of transgenic lines under transgenic screen house conditions revealed that line # A16 exhibited on par agronomic performance against its non-transgenic counterpart under normal conditions. Under drought, non-transgenic ADT 43 showed >20% reduction in the total number of spikelets per panicle, whereas transgenic line # A16 registered only a 2% reduction. Non-transgenic ADT 43 recorded 80% yield reduction under drought, whereas line # A16 recorded only 54% yield loss. The above results demonstrated the effectiveness of controlled expression of DREB1A in regulating dehydration responses in rice.

Transgenic chickpea (Cicer arietinum L.) harbouring AtDREB1a are physiologically better adapted to water deficit

BackgroundChickpea (Cicer arietinum L.) is the second most widely grown pulse and drought (limiting water) is one of the major constraints leading to about 40–50% yield losses annually. Dehydration responsive element binding proteins (DREBs) are important plant transcription factors that regulate the expression of many stress-inducible genes and play a critical role in improving the abiotic stress tolerance. Transgenic chickpea lines harbouring transcription factor, Dehydration Responsive Element-Binding protein 1A from Arabidopsis thaliana (AtDREB1a gene) driven by stress inducible promoter rd29a were developed, with the intent of enhancing drought tolerance in chickpea. Performance of the progenies of one transgenic event and control were assessed based on key physiological traits imparting drought tolerance such as plant water relation characteristics, chlorophyll retention, photosynthesis, membrane stability and water use efficiency under water stressed conditions.ResultsFour transgenic chickpea lines harbouring stress inducible AtDREB1a were generated with transformation efficiency of 0.1%. The integration, transmission and regulated expression were confirmed by Polymerase Chain Reaction (PCR), Southern Blot hybridization and Reverse Transcriptase polymerase chain reaction (RT-PCR), respectively. Transgenic chickpea lines exhibited higher relative water content, longer chlorophyll retention capacity and higher osmotic adjustment under severe drought stress (stress level 4), as compared to control. The enhanced drought tolerance in transgenic chickpea lines were also manifested by undeterred photosynthesis involving enhanced quantum yield of PSII, electron transport rate at saturated irradiance levels and maintaining higher relative water content in leaves under relatively severe soil water deficit. Further, lower values of carbon isotope discrimination in some transgenic chickpea lines indicated higher water use efficiency. Transgenic chickpea lines exhibiting better OA resulted in higher seed yield, with progressive increase in water stress, as compared to control.ConclusionsBased on precise phenotyping, involving non-invasive chlorophyll fluorescence imaging, carbon isotope discrimination, osmotic adjustment, higher chlorophyll retention and membrane stability index, it can be concluded that AtDREB1a transgenic chickpea lines were better adapted to water deficit by modifying important physiological traits. The selected transgenic chickpea event would be a valuable resource that can be used in pre-breeding or directly in varietal development programs for enhanced drought tolerance under parched conditions.

Independent overexpression of OsRab16A and AtDREB1A exhibit enhanced drought tolerance in transgenic aromatic rice variety Pusa Sugandhi 2

Rice (Oryza sativa L.), the staple food in South-East Asian countries, is a water-intensive crop so that its productivity is highly affected by drought, the most severe environmental stress factor. We report here the development of transgenic rice via overexpression of full length OsRab16A (driven by endogenous promoter) or AtDREB1A driven by rd29A promoter in the background of the aromatic indica rice cultivar, Pusa Sugandhi 2 (PS2) through biolistic transformation. Both the transgenes were stably introgretted in T2 generations as indicated by the results of polymerase chain reaction (PCR). Reverse transcriptase (RT)-polymerase chain reaction (PCR) analyses showed that the expression of both the transgenes was induced by drought stress in all the T2 transgenic plants examined, which were highly tolerant to water deficit stress during both the vegetative and reproductive stages without their morphological or agronomic traits being affected. Protein immunoblot analysis with Rab16A (= anti dehydrin) antiserum showed detectable accumulation of Rab16A protein in the transgenics in response to drought stress and exogenous abscisic acid (ABA) application. The physiological studies revealed that the expression of the transgene under drought stress in T2 transgenics was associated with lesser shoot or root length inhibition, improved leaf relative water content, stable maintenance of chlorophyll, lesser hydrogen peroxide accumulation with higher catalase activity and an increased accumulation of the osmoprotectant proline, as compared with the wild type (WT) plants. The transgenic plants also showed significantly higher filled grain, spikelet fertility and grain yield under stressed conditions. All these findings highlighted the tremendous potentiality of both OsRab16A and AtDREB1A in conferring drought tolerance without affecting the normal phenotype and physiology of the plants. To our knowledge, this is the first report on the development of a drought-tolerant transgenic aromatic rice variety through overexpression of genes, one involved in ABA-dependent pathway (OsRab16A), while the other in ABA-independent pathway (AtDREB1A) of stress tolerance, thereby highlighting the significance of both the pathways in drought tolerance of the aromatic rice varieties.

Heterologous expression of the ThIPK2 gene enhances drought resistance of common wheat

ThIPK2 is an inositol polyphosphate kinase gene cloned from Thellungiella halophila that participates in diverse cellular processes. Drought is a major limiting factor in wheat (Triticum aestivum L.) production. The present study investigated whether the application of the ThIPK2 gene could increase the drought resistance of transgenic wheat. The codon-optimized ThIPK2 gene was transferred into common wheat through Agrobacterium-mediated transformation driven by either a constitutive maize ubiquitin promoter or a stress-inducible rd29A promoter from Arabidopsis. Molecular characterization confirmed the presence of the foreign gene in the transformed plants. The transgenic expression of ThIPK2 in wheat led to significantly improve drought tolerance compared to that observed in control plants. Compared to the wild type (WT) plants, the transgenic plants showed higher seed germination rates, better developed root systems, a higher relative water content (RWC) and total soluble sugar content, and less cell membrane damage under drought stress conditions. The expression profiles showed different expression patterns with the use of different promoters. The codon-optimized ThIPK2 gene is a candidate gene to enhance wheat drought stress tolerance by genetic engineering.

Overexpression of ABA Receptor PYL10 Gene Confers Drought and Cold Tolerance to Indica Rice.

Abscisic acid (ABA) plays versatile functions in regulating plant development and tolerance to various biotic and abiotic stresses. Towards elucidating the functions of one of the ABA receptors (ABARs) in rice, OsPYL10 was cloned from drought tolerant rice cv. Nagina 22 and was overexpressed under stress inducible RD29A promoter in a mega rice variety MTU1010 by using Agrobacterium mediated genetic transformation. Four single copy transgenic lines selected based on Southern blot analysis were used for physiological and molecular analysis. PYL10 receptor appears to regulate its ligand ABA accumulation as PYL10 overexpressing transgenics accumulated 2–3.3-fold higher levels of ABA than that of WT in flag leaf at anthesis under non-stress conditions. The enhanced accumulation of ABA was associated with enhanced expression of genes for ABA biosynthesis viz., ZEP1, NCED1, NCED2, NCED3, and NCED4 in transgenics than in WT plants. At seedling stage, PYL10 transgenics showed significantly higher survival rate under cold stress as compared with WT plants. qRT-PCR analysis showed that expression levels of cold responsive genes viz., DREB1F, MYB3R2, TPP1, COR410, DEHYDRIN, and LEA3 were significantly higher in PYL10 overexpressing transgenic lines as compared to WT plants under cold stress. PYL10 transgenic and WT plants grown in the same pot were subjected to -80 kPa drought stress and recovery treatments at vegetative and reproductive stages. At vegetative stage drought stress, three overexpressing lines showed significantly higher grain yield (40–58%) and at reproductive stage drought stress one of these overexpression lines showed two-fold higher grain yield than that of WT plants. Excised leaf water loss analysis showed that PYL10 transgenic lost about 20% less water than WT plants. At reproductive stage, OsPYL10 transgenic maintained higher RWC, membrane stability index, chlorophyll content, and accumulated lower amount of MDA and H2O2 as compared with WT plants. qRT-PCR analysis showed that expression levels of RAB16, Dehydrin, LEA3, and ABA45 were higher in PYL10 transgenics as compared with WT plants under drought stress. Thus, overall results showed that OsPYL10 overexpression has potential to improve both drought and cold stress tolerance of indica rice.

Ectopic expression of AtSOC1 gene driven by the inducible promoter rd29A, causes early flowering in Chrysanthemum

Suppressor of Overexpression of Constans1 of Arabidopsis (AtSOC1) plays an important role in floral induction and development as one of the flowering pathway integrators. rd29A is a stress inducible promoter. To obtain a new chrysanthemum germplasm with an inducible flowering time, we succeeded in making transgenic chrysanthemum plants carrying AtSOC1 genes driven by the rd29A promoter via Agrobacterium-mediated transformation. Molecular characterization confirmed the stable integration of the introduced gene cassette in the transformants. The result showed that AtSOC1 expression was elevated to varying extents in different transgenic lines. One of the lines showed an obvious phenotype of early flowering, which was induced by drought stress. These results indicated that AtSOC1, driven by the rd29A promoter, can regulate the flowering time when drought stress treatments are applied to chrysanthemum.