Generation Of Transgenic Plants Research Articles

Agrobacterium-Mediated Transformation of Setaria viridis, a Model System for Cereals and Bioenergy Crops.

Setaria viridis is an emerging model system for the genetic and molecular characterization of cereals and bioenergy crops. Here, we describe a detailed procedure for genetic transformation of the S. viridis accession line ME034V-1. This method utilizes callus generated from mature seeds for infection with Agrobacterium tumefaciens strain AGL1 to regenerate hygromycin-resistant stable transgenic plants. It takes approximately 7 weeks to generate callus from mature seeds, 11-17 weeks from infection to the regeneration of transgenic lines, and an additional 3-4 weeks for plant growth in the greenhouse for seed collection. The protocol as presented consistently results in transformation frequency of approximately 25% for the generation of transgenic plants, with fewer escapes and higher survivability in soil for optimal seed collection. © 2021 Donald Danforth Plant Science Center. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of S. viridis (Accession ME034V-1) callus from mature seeds Basic Protocol 2: Agrobacterium-mediated transformation of callus to generate transgenic plants Basic Protocol 3: Plantlet transplantation in soil, plant growth in greenhouse, and seed collection Support Protocol: Preparation of Agrobacterium culture for infection.

Exploitation of Novel Bt ICPs for the Management of Helicoverpa armigera (Hübner) in Cotton (Gossypium hirsutum L.): A Transgenic Approach

Cotton is a commercial crop of global importance. The major threat challenging the productivity in cotton has been the lepidopteron insect pest Helicoverpa armigera or cotton bollworm which voraciously feeds on various plant parts. Biotechnological interventions to manage this herbivore have been a universally inevitable option. The advent of plant genetic engineering and exploitation of Bacillus thuringiensis (Bt) insecticidal crystal proteins (ICPs) marked the beginning of plant protection in cotton through transgenic technology. Despite phenomenal success and widespread acceptance, the fear of resistance development in insects has been a perennial concern. To address this issue, alternate strategies like introgression of a combination of cry protein genes and protein-engineered chimeric toxin genes came into practice. The utility of chimeric toxins produced by domain swapping, rearrangement of domains, and other strategies aid in toxins emerging with broad spectrum efficacy that facilitate the avoidance of resistance in insects toward cry toxins. The present study demonstrates the utility of two Bt ICPs, cry1AcF (produced by domain swapping) and cry2Aa (produced by codon modification) in transgenic cotton for the mitigation of H. armigera. Transgenics were developed in cotton cv. Pusa 8–6 by the exploitation of an apical meristem-targeted in planta transformation protocol. Stringent trait efficacy-based selective screening of T1 and T2 generation transgenic plants enabled the identification of plants resistant to H. armigera upon deliberate challenging. Evaluation of shortlisted events in T3 generation identified a total of nine superior transgenic events with both the genes (six with cry1AcF and three with cry2Aa). The transgenic plants depicted 80–100% larval mortality of H. armigera and 10–30% leaf damage. Molecular characterization of the shortlisted transgenics demonstrated stable integration, inheritance and expression of transgenes. The study is the first of its kind to utilise a non-tissue culture-based transformation strategy for the development of stable transgenics in cotton harbouring two novel genes, cry1AcF and cry2Aa for insect resistance. The identified transgenic events can be potential options toward the exploitation of unique cry genes for the management of the polyphagous insect pest H. armigera.

Genes with Cold Shock Domain from Eutrema salsugineum (Pall.) for Generating a Cold Stress Tolerance in Winter Rape (Brassica napus L.) Plants

The increased demand in vegetable oil for food purposes and high-protein feed for livestock and poultry encourages producers to expand the production of various oil crops, while occupying rather cold agroclimatic zones. Improved cold and frost resistance of cultivated crops would significantly increase the yield and expand the range of rape cultivation in a number of cold climate regions. Nine transgenic lines of winter rape containing genes encoding proteins with a cold shock domain (CspA и EsCSDP3) were obtained as a result of Agrobacterium transformation. In total, 260 explants were involved in transformation of rape using pBI121-CSPA-plant, with a transformation efficiency of 2.3%; among 750 explants using the pBI-EsCSDP3 construction, the efficiency was 0.4%. As a result of the studies, it was shown that the expression of the new gene Escsdp3 from the plant of Eutrema salsugineum was able to increase the cold and frost resistance of plants as effectively as the cspa gene from E. coli, which is classically used for this purpose. The cold resistance analysis of T1 transgenic plants generation revealed four cold resistant winter rape lines (three lines with the cspA-plant gene and one line with the Escsdp3 gene). The transfer of Escsdp3 and cspA-plant genes into winter rape plants led to a significant increase in frost resistance of plants. Two winter rapeseed lines were resistant to freezing (with the cspA-plant gene and with the Escsdp3 gene). Non-hardened transgenic plants remained viable after 24 h of exposure to negative temperatures up to −5 °C, and plants that passed through the hardening stage survived after freezing at −16 °C.

Digital PCR (dPCR) and qPCR mediated determination of transgene copy number in the forage legume white clover (Trifolium repens).

Obtaining data on transgene copy number is an integral step in the generation of transgenic plants. Techniques such as Southern blot, segregation analysis, and quantitative PCR (qPCR) have routinely been used for this task, in a range of species. More recently, use of Digital PCR (dPCR) has become prevalent, with a measurement accuracy higher than qPCR reported. Here, the relative merits of qPCR and dPCR for transgene copy number estimation in white clover were investigated. Furthermore, given that single copy reference genes are desirable for estimating gene copy number by relative quantification, and that no single-copy genes have been reported in this species, a search and evaluation of suitable reference genes in white clover was undertaken. Results demonstrated a higher accuracy of dPCR relative to qPCR for copy number estimation in white clover. Two genes, Pyruvate dehydrogenase (PDH), and an ATP-dependent protease, identified as single-copy genes, were used as references for copy number estimation by relative quantification. Identification of single-copy genes in white clover will enable the application of relative quantification for copy number estimation of other genes or transgenes in the species. The results generated here validate the use of dPCR as a reliable strategy for transgene copy number estimation in white clover, and provide resources for future copy number studies in this species.

Isolation, Characterization, and Optimization of Protease‑Producing Bacterium Bacillus thuringiensis from Paddy Field Soil

Background: The ubiquitous proteases that are commonly found in all living organisms play an important role in cell growth and cell differentiation. The bacterium Bacillus thuringiensis (Bt) produces delta-endotoxins that exhibit toxic properties against various insecticides and has demonstrated its potency and safety as a biopesticide agent for decades. The Bt protein includes vegetative, insecticidal, and crystal proteins that exhibit highly toxicants against immature insects (larvae). Objectives: The aim of this research was to use Bt as an alternative to chemical insecticides, and the source of Bt genes aids in the development of a resistant transgenic plant that improves not only productivity but also shift life. Materials and Methods: In the present study, bacterium Bt was isolated from various paddy files around the Hunsur region, Karnataka. The isolated bacteria show a potent protease activity on skim agar plates. Morphology, colony assay, and biochemical characterization were performed for the characteristic properties of bacteria. Further, 16S ribosomal RNA partial sequencing was carried out to identify the specific species of Bacillus. Results: Among nine samples from different paddy soils, three Bacillus isolates SAL-P1, SAL-P2, and SAL-P3 are the major dominant colonies which were streaked onto the fresh skim milk agar plates, out of which SAL-Pl shows an abundant growth and production of an enzyme at pH 7.0, 37°C, and 48 h, respectively. The study also shows the optimum condition of temperature, carbon, nitrogen source, pH for growth, as well as for biomass production. Conclusion: The results of this study confirm the significance of continuous exploration of new Bt strains from different ecological regions that could be more useful for Bt-based bioformulations and the generation of transgenic plants. Furthermore, the growth and biomass production of Btg (isolated from paddy soil) and Bti (reference strain) were found to be identical.

Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants.

SUMMARYCowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium‐mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium‐mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas‐mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies.

Regulated promoters applied to plant engineering: an insight over promising soybean promoters under biotic stress and their cis-elements

Promoters are upstream gene regulatory sequences recognized by transcription factors (TFs) involved in controlling transcription initiation and progression. For modern crop improvement, the design of efficient gene constructs relies on promoter efficiency, tissue specificity, and other characteristics that allow the introgression of agronomically relevant traits to overcome biotic and abiotic stresses. Several constitutive viral promoters, such as pCaMV35S, remain widely employed in the transgenic plant generation, but their indiscriminate use leads to gene silencing triggering and metabolic penalties impacting plant fitness. The identification and functional characterization of plant-derived promoters can unveil alternatives to commonly used non-homologous promoters; however, knowledge over them remains limited, especially for crops. This review summarizes plant promoters used to drive foreign gene expression in homologous and heterologous systems, focusing on inducible soybean promoters from genes upregulated by different biotic stresses. Analyses of these soybean promoters revealed 22 coincident cis-acting elements that can be used for synthetic engineering promoters responsive to multiple biotic stresses and, therefore, efficiently drive gene expression, conferring desirable traits in transgenic soybean. In addition, we also revisited commercial and protected promoters to provide an update on soybean promoters and gain new insights into superior crops’ development.

Construction of a new plant expression vector and the development of maize germplasm expressing the Aspergillus ficuum phytase gene PhyA2

Phytases, which belong to a special category of orthophosphoric monoester phosphohydrolases, degrade inositol hexaphosphate to produce lower-grade inositol phosphate derivatives and inorganic phosphate. Thus, phytases may improve phosphorus utilization, eliminate the anti-nutrient properties of phytic acid, and mitigate environmental pollution due to phosphorus contamination. In this study, we constructed a new root-specific expression vector by inserting the Aspergillus ficuum phytase gene PhyA2 into pCAMBIA3301-ZmGLU1P-Nos. The subsequent molecular analysis confirmed that six T4 generation transgenic plants carried and expressed PhyA2. A quantitative real-time PCR analysis indicated PhyA2 was highly expressed in the transgenic roots. Additionally, the phytase activity was 10.9-fold higher in the transgenic roots (peak activity of 5.432 U/g) than in the control roots. Moreover, compared with the control rhizosphere, the organic phosphorus content in the rhizosphere of the transgenic plants decreased significantly (by up to 5.21 mg/kg). An agronomic trait analysis indicated that PhyA2 expression can increase maize seed weight by up to 25.8 g. Therefore, the integration of PhyA2 into the maize genome can enhance the ability of maize plants to use the phosphorus compounds in soil, while also improving the plant growth status and increasing the seed yield.

Agrobacterium tumefaciens-mediated in planta transformation strategy for development of transgenics in cotton (Gossypium hirsutum L.) with GFP as a visual marker.

Cotton (Gossypium hirsutum L.), a mercantile crop plant, is grown worldwide for fiber and seed oil. As with other economically important crops, cotton is bogged down with many biotic and abiotic stress factors. Towards this,genetic engineering offers numerous protocols to engineer plantsfor better resilience. However, recalcitrance of cotton to planttissue culture has been the major constraint for successfulin vitro regeneration. Hence,alternate methods that evade tissue culture regeneration have been envisaged. Non tissue culture-based in planta transformation strategies are in vogue due to amenability and ease in the generation of transgenic plants. In the present study, we demonstrate the utility of an in planta transformation protocol and establishment of a stringent selection agent-based screening for the identificationof transgenics. The genotype independent nature of the protocol was validated in cotton cv. Pusa 8-6 using GFP. Preliminary transformation efficiency of 28% was achieved with a screening efficiency of 20% in the presence of hygromycin. The proof of T-DNA integration by various molecular and expression analysis in T1 and T2 generations proved that this technique can be employed to generate transgenic cotton.

Engineering Chickpea Variety HC-1 with OsRuvB Gene for Salt Stress Tolerance

Background: Salinity is a major problem worldwide and is increasing day by day. Salt stress causes severe yield losses in crop plants and the damages in chickpea are up to 100%. To overcome these losses, the present study was undertaken to develop transgenic chickpea plants (var. HC-1) carrying OsRuvB gene for salt stress tolerance. Methods: Transgenic chickpea plants harboring OsRuvB gene were developed using Agrobacterium-mediated transformation. T0 putative transgenic chickpea plants were screened for the presence of OsRuvB gene through PCR using gene specific primers. The stable integration and copy number of transgene in transgenic chickpea plants were confirmed through Southern hybridization and qRT-PCR. T1 generation transgenic chickpea plants were screened for the presence of OsRuvB gene using direct PCR (Phire Direct PCR kit). Result: PCR-based screening of putative transformants using gene-specific primers showed a transformation frequency of 17%. Southern blot and real-time PCR analysis revealed stable and single-copy insertion. In T1 generation a total of 74 plants (out of 170) showed the presence of OsRuvB gene. The engineered lines developed in the present investigation can be further undertaken to develop transgenic chickpea plants for salt stress tolerance.

Improving crops genome through genetic engineering of the key metabolic pathways

Soybean loss due to pests and pathogens is a serious problem worldwide. Soybean producers have few options to manage diseases caused by general pathogens where major genes for full resistance have not been discovered. The innate defense of soybean plants could be enhanced by improving content and composition of lignin by genetic engineering of the phenylpropanoid pathway. We used a novel technique of germ-line genetic transformation of soybean plants via natural pollen tubes as vectors. This technique uses Agrobacterium tumefaciens to mediate transfer of genes of interest to the zygote to introduce the key lignification genes (PtMYB4, PAL5, F5H, CAD1) into soybean genome. We observed 5.6% average transformation efficiency in the first generation of transgenic plants and in the second generation the presence of the transgene constructs was confirmed in more than 50% (for CsVMV/PtMYB4sens, 35SVTM/PAL5, C4H/F5H, CsVMV/CAD1 constructs) transgenic soybean lines. We confirmed the expression of the introduced genes at transcriptional level using RT-PCR and Northern blot. Functional analysis using lignin content determination and the activity of PAL5 and CAD1 enzymes demonstrated that the transgenes perform their function in planta. The proposed technique is effective and inexpensive and can be used to create novel stress and disease resistant soybean genotypes.

Application of Exogenous dsRNAs-induced RNAi in Agriculture: Challenges and Triumphs.

In recent years, RNA interference (RNAi) machinery has widely been explored by plant biologists for its potential applications in disease management, plant development, and germplasm improvement. RNAi-based technologies have mainly been applied in the form of transgenic plant generation and host-induced-gene-silencing (HIGS). However, the approval of RNAi-based transgenic plants has always been challenging due to the proclaimed concerns surrounding their impacts on human health and the environment. Lately, exogenous applications of double-stranded RNAs (dsRNAs), short interfering RNAs (siRNAs), and hairpin RNAs (hpRNAs) has emerged as another technology that could be regarded as more eco-friendly, sustainable, and publicly acceptable than genetic transformation. Inside the plant cell, dsRNAs can undergo several steps of processing, which not only triggers RNAi machinery but may also involve transitive and systemic silencing, as well as epigenetic modifications. Therefore, along with the considerations of proper exogenous applications of dsRNAs, defining their final destination into plant cells is highly relevant. In this review, we highlighted the significance of several factors that affect dsRNA-induced gene silencing, the fate of exogenous dsRNAs in the plant cell, and the challenges surrounding production technologies, cost-effectiveness, and dsRNAs stability under open-field conditions. This review also provided insights into the potential applications of exogenous dsRNAs in plant protection and crop improvement.

Efficient targeted mutagenesis in allotetraploid sweet basil by CRISPR/Cas9.

Sweet basil (Ocimum basilicum) is an economically important herb and its global production is threatened by basil downy mildew caused by the obligate biotrophic oomycete Peronospora belbahrii. Effective tools are required for functional understanding of its genes involved in synthesis of valuable secondary metabolites in essential oil and disease resistance, and breeding for varieties with improved traits. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene editing technology has revolutionized crop breeding and functional genomics. The applicability and efficacy of this genomic tool in the allotetraploid sweet basil were tested by editing a potential susceptibility (S) gene ObDMR1, the basil homolog of Arabidopsis DMR1 (Downy Mildew Resistant 1) whose mutations conferred nearly complete resistance against Arabidopsis downy mildew pathogen, Hyaloperonospora arabidopsidis. Two single guide RNAs targeting two different sites of the ObDMR1 coding sequence were designed. A total of 56 transgenic lines were obtained via Agrobacterium‐mediated stable transformation. Mutational analysis of 54 T0 transgenic lines identified 92.6% lines carrying mutations at target 1 site, while a very low mutation frequency was detected at target 2 site. Deep sequencing of six T0 lines revealed various mutations at target 1 site, with a complete knockout of all alleles in one line. ObDMR1 homozygous mutant plants with some being transgene free were identified from T1 segregating populations. T2 homozygous mutant plants with 1‐bp frameshift mutations exhibited a dwarf phenotype at young seedling stage. In summary, this study established a highly efficient CRISPR/Cas9‐mediated gene editing system for targeted mutagenesis in sweet basil. This system has the capacity to generate complete knockout mutants in this allotetraploid species at the first generation of transgenic plants and transgene‐free homozygous mutants in the second generation. The establishment of this system is expected to accelerate basil functional genomics and breeding.

Establishment of a plant tissue culture system and genetic transformation for agronomic improvement of Indonesian black rice (Oryza sativa L.)

Indonesian black rice has the potential to be developed as a functional food due to their high anthocyanin content and other nutritional benefits such as enhanced Fe and amino acid composition. Black rice productivity, however, is low due to its long harvesting time, caused by late heading date, and therefore poor yield. These negative traits in black rice may be improved using conventional and modern approaches by manipulating genes of interest. As a tool to study functional genomics in black rice, genetic transformation was studied. A suitable in vitro regeneration method, previously unavailable for Indonesian black rice cultivars, first had to be established as a key prerequisite for genetic transformation. The purpose of this study therefore was to establish an efficient method for culturing Indonesian black rice cultivars and subsequently to use the Hd3a flowering gene under the RPP16 promoter as a relevant gene to improve the flowering period with either in vitro or in planta transformation methods. We found that the in vitro regeneration method established was able to generate fertile black rice plants and furthermore enabled the generation of transgenic plants. T0 transgenics harboring the Hd3a gene driven by the RPP16 promoter yielded extremely early flowering, dwarfed, and low-yielding plants, but we obtained a normal phenotype with early flowering in the T1 generation. In vitro transformation was more efficient in terms of time taken and number of positive transformants than in planta transformation for genetic transformation of Indonesian black rice by Agrobacterium-mediated transformation. Indonesian black rice requires a specific temperature range of 30–32 oC during the tissue culture phase for successful genetic transformation.

Plant Fatty Acid Desaturases: Role in the Life of Plants and Biotechnological Potential

Fatty acid desaturases perform a key role in the maintenance of cell membrane homeostasis. They convert fatty acids of membrane lipids from saturated to unsaturated. The modulation of fatty acid unsaturation in cell membranes is one of the mechanisms of plant adaptation to traumatic environmental factors, both biotic and abiotic. The study of the mechanisms of the functioning of desaturases, as well as their localization and characteristics of their gene expression, carry out a fundamental role in our understanding of the processes of plant adaptation. In addition to their fundamental importance, such studies have an applied character. They will expand the potential of desaturases and put in place the foundations of a modern generation of transgenic plants, including those derived from genome-editing technologies. As a result, these studies deliver a broad benefit to humanity in the creation of stress-resistant crops or the biofortification of agricultural crops.

Enhancement of glyphosate tolerance in rice (Oryza sativa L.) through mutation induction in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase)

Generation of an herbicide-tolerant cultivar could be an important goal in rice breeding program. Therefore, a direct mutagenesis approach (megaprimer technique) was employed to generate the EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) mutant version called epsps in which glycine 96 was replaced by alanine. Then both EPSPS and epsps genes were separately cloned into pET22b system following transformation of E. coli BL21. The transformed bacteria showed a higher expression level of EPSPS and epsps extending their survival in presence of high levels of glyphosate. Interestingly, the bacteria harboring pET22b:epsps could grow in the medium containing 18 mM glyphosate. Subsequently, Hashemi rice cultivar was used to generate two types of transgenic rice including plants harboring either EPSPS (35S:EPSPS) or epsps (35S:epsps). Molecular analysis was performed on T3 putative transgenic plants so as to confirm the presence and expression of transgenes. The results demonstrated that 35S:epsps is actively and stably transcribed in T3 transgenic plants and developed a considerable tolerance towards glyphosate in comparison with the 35S:EPSPS plants. When compared to non-transgenic plants, the epsps mutant plants accumulated higher levels of chlorophyll and carotenoid in association with less shikimic acid, which are considered as some indicators of glyphosate side effects. Moreover, flavonoid accumulation was accompanied by enhanced levels of alkaloids and lower levels of proline in 35S:epsps plants; indicating that shikimic acid pathway is well functioning and transgenic plants especially 35S:epsps experienced less stress. Therefore, for the first time, rice was successfully transformed by 35S:epsps resulting in generation of transgenic plants with a considerable level of glyphosate tolerance.

Tomato glycosyltransferase Twi1 plays a role in flavonoid glycosylation and defence against virus.

BackgroundSecondary metabolites play an important role in the plant defensive response. They are produced as a defence mechanism against biotic stress by providing plants with antimicrobial and antioxidant weapons. In higher plants, the majority of secondary metabolites accumulate as glycoconjugates. Glycosylation is one of the commonest modifications of secondary metabolites, and is carried out by enzymes called glycosyltransferases.ResultsHere we provide evidence that the previously described tomato wound and pathogen-induced glycosyltransferase Twi1 displays in vitro activity toward the coumarins scopoletin, umbelliferone and esculetin, and the flavonoids quercetin and kaempferol, by uncovering a new role of this gene in plant glycosylation. To test its activity in vivo, Twi1-silenced transgenic tomato plants were generated and infected with Tomato spotted wilt virus. The Twi1-silenced plants showed a differential accumulation of Twi1 substrates and enhanced susceptibility to the virus.ConclusionsBiochemical in vitro assays and transgenic plants generation proved to be useful strategies to assign a role of tomato Twi1 in the plant defence response. Twi1 glycosyltransferase showed to regulate quercetin and kaempferol levels in tomato plants, affecting plant resistance to viral infection.

Agrobacterium tumefaciens-mediated transformation of a hevein-like gene into asparagus leads to stem wilt resistance.

Asparagus stem wilt, is a significant and devastating disease, typically leading to extensive economic losses in the asparagus industry. To obtain transgenic plants resistant to stem wilt, the hevein-like gene, providing broad spectrum bacterial resistance was inserted into the asparagus genome through Agrobacterium tumefaciens-mediated transformation. The optimal genetic transformation system for asparagus was as follows: pre-culture of embryos for 2 days, inoculation using a bacterial titre of OD600 = 0.6, infection time 10 min and co-culturing for 4 days using an Acetosyringone concentration of 200 μmol/L. Highest transformation frequencies reached 21% and ten transgenic asparagus seedlings carrying the hevein-like gene were identified by polymerase chain reaction. Moreover, integration of the hevein-like gene in the T1 generation of transgenic plants was confirmed by southern blot hybridization. Analysis showed that resistance to stem wilt was enhanced significantly in the transgenic plants, in comparison to non- transgenic plants. The results provide additional data for genetic improvement and are of importance for the development of new disease-resistant asparagus varieties.

The Interplay among Polyamines and Nitrogen in Plant Stress Responses.

The interplay between polyamines (PAs) and nitrogen (N) is emerging as a key factor in plant response to abiotic and biotic stresses. The PA/N interplay in plants connects N metabolism, carbon (C) fixation, and secondary metabolism pathways. Glutamate, a pivotal N-containing molecule, is responsible for the biosynthesis of proline (Pro), arginine (Arg) and ornithine (Orn) and constitutes a main common pathway for PAs and C/N assimilation/incorporation implicated in various stresses. PAs and their derivatives are important signaling molecules, as they act largely by protecting and preserving the function/structure of cells in response to stresses. Use of different research approaches, such as generation of transgenic plants with modified intracellular N and PA homeostasis, has helped to elucidate a plethora of PA roles, underpinning their function as a major player in plant stress responses. In this context, a range of transgenic plants over-or under-expressing N/PA metabolic genes has been developed in an effort to decipher their implication in stress signaling. The current review describes how N and PAs regulate plant growth and facilitate crop acclimatization to adverse environments in an attempt to further elucidate the N-PAs interplay against abiotic and biotic stresses, as well as the mechanisms controlling N-PA genes/enzymes and metabolites.

Overexpression of the arginine decarboxylase gene promotes the symbiotic interaction Medicago truncatula-Sinorhizobium meliloti and induces the accumulation of proline and spermine in nodules under salt stress conditions

Legumes have the capacity to fix nitrogen in symbiosis with soil bacteria known as rhizobia by the formation of root nodules. However, nitrogen fixation is highly sensitive to soil salinity with a concomitant reduction of the plant yield and soil fertilization. Polycationic aliphatic amines known as polyamines (PAs) have been shown to be involved in the response to a variety of stresses in plants including soil salinity. Therefore, the generation of transgenic plants overexpressing genes involved in PA biosynthesis have been proposed as a promising tool to improve salt stress tolerance in plants. In this work we tested whether the modulation of PAs in transgenic Medicago truncatula plants was advantageous for the symbiotic interaction with Sinorhizobium meliloti under salt stress conditions, when compared to wild type plants. Consequently, we characterized the symbiotic response to salt stress of the homozygous M. truncatula plant line L-108, constitutively expressing the oat adc gene, coding for the PA biosynthetic enzyme arginine decarboxylase, involved in PAs biosynthesis. In a nodulation kinetic assay, nodule number incremented in L-108 plants under salt stress. In addition, these plants at vegetative stage showed higher nitrogenase and nodule biomass and, under salt stress, accumulated proline (Pro) and spermine (Spm) in nodules, while in wt plants, the accumulation of glutamic acid (Glu), γ-amino butyric acid (GABA) and 1-aminocyclopropane carboxylic acid (ACC) (the ethylene (ET) precursor) were the metabolites involved in the salt stress response. Therefore, overexpression of oat adc gene favours the symbiotic interaction between plants of M. truncatula L-108 and S. meliloti under salt stress and the accumulation of Pro and Spm, seems to be the molecules involved in salt stress tolerance.