Biotechnological Advances to Improve Abiotic Stress Tolerance in Crops.

The major challenges that agriculture is facing in the twenty-first century are increasing droughts, water scarcity, flooding, poorer soils, and extreme temperatures due to climate change. However, most crops are not tolerant to extreme climatic environments. The aim in the near future, in a world with hunger and an increasing population, is to breed and/or engineer crops to tolerate abiotic stress with a higher yield. Some crop varieties display a certain degree of tolerance, which has been exploited by plant breeders to develop varieties that thrive under stress conditions. Moreover, a long list of genes involved in abiotic stress tolerance have been identified and characterized by molecular techniques and overexpressed individually in plant transformation experiments. Nevertheless, stress tolerance phenotypes are polygenetic traits, which current genomic tools are dissecting to exploit their use by accelerating genetic introgression using molecular markers or site-directed mutagenesis such as CRISPR-Cas9. In this review, we describe plant mechanisms to sense and tolerate adverse climate conditions and examine and discuss classic and new molecular tools to select and improve abiotic stress tolerance in major crops.

Reduction in Salt Stress Due to the Action of Halophilic Bacteria That Promote Plant Growth in Solanum lycopersicum.

Soil salinity is one of the most important factors reducing agricultural productivity worldwide. Halophilic plant growth-promoting bacteria (H-PGPB) represent an alternative method of alleviating saline stress in crops of agricultural interest. In this study, the following halophilic bacteria were evaluated: Bacillus sp. SVHM1.1, Halomonas sp. SVCN6, Halomonas sp. SVHM8, and a consortium. They were grown under greenhouse conditions in Solanum lycopersicum at different salinity concentrations in irrigation water (0, 20, 60, and 100 mM NaCl) to determine the effects on germination, fruit quality, yield, and concentration of osmoprotectors in plant tissue. Our results demonstrate the influence of halophilic bacteria with the capacity to promote plant growth on the germination and development of Solanum lycopersicum at higher salinity levels. The germination percentage was improved at the highest concentration by the inoculated treatments (from 37 to 47%), as were the length of the radicle (30% at 20 mM) and plumule of the germinated seed, this bacterium also increased the weight of the plumule (97% at 100 mM). They also improved the yield. The dry weight of the plant, in addition to having an influence on the quality of the fruit and the concentration of osmoprotectors (Bacillus sp. SVHM 1.1) had the greatest effect on fruit yield (1.5 kg/plant at 20 mM), by the otherhand, Halomonas sp. SVHM8 provided the best fruit quality characteristics at 100 mM. According to the above results, the efficiency of halophilic PGPB in the attenuation of salt stress in Solanum lycopersicum has been proven.

Identification of Halophilic and Halotolerant Bacteria from the Root Soil of the Halophyte Sesuvium verrucosum Raf.

Soil salinity is a condition that limits crop growth and productivity, and soil-dwelling bacteria from halophytic plant roots may be a viable strategy to cope with low productivity due to salt stress. Halophilic and halotolerant bacteria of the root soil of Sesuvium verrucosum were analyzed in this study as there is little evidence regarding its associated microbiology. Soil was sampled from the roots of Sesuvium verrucosum to obtain the cultivable bacteria. Their morphological characteristics were identified and they were molecularly identified by the 16S sequence. The growth capacity of the bacteria was determined at different levels of pH and salinity, and several growth promotion characteristics were identified, such as phosphorus solubilization, indole acetic acid production by the tryptophan-dependent (AIAt) and tryptophan-independent (IAA) pathways, ammonium production from organic sources, solubilization of carbonates, and zinc and sodium capture capacity. In addition, the bacteria that presented the best characteristics for germination variables of Solanum lycopersicum were evaluated. A total of 20 bacteria from root soil of Sesuvium verrucosum Raf. belonging to the phyla Proteobacteria (50%), Firmicutes (45%) and Actinobacteria (5%) were identified, with each one having different morphological characteristics. Among the bacterial isolates, 45% had the ability to resist different levels of salinity and pH, ranging from 0 to 20% of NaCl, and pH between 5 and 11. Moreover, these bacteria had the capacity to solubilize carbonates, phosphorus and zinc, capture sodium, produce ammonium from organic substrates and IAA (indole acetic acid), and promote enzymatic activity of amylases, proteases, lipases and cellulases. The bacteria evaluated on the germination of Solanum lycopersicum had an influence on germination at different salinity levels, with greater influence at 100 mM NaCl. This demonstrated that halophilic bacteria belonging to the rhizosphere of Sesuvium verrucosum have the ability to promote growth in extreme salinity conditions, making them candidates for the recovery of productivity in saline soils.

A Versatile Peroxidase from the Fungus Bjerkandera adusta Confers Abiotic Stress Tolerance in Transgenic Tobacco Plants.

White-rot fungi are efficient lignin degraders due to the secretion of lignin peroxidase, manganese peroxidase, laccase, and versatile peroxidase (VP) on decayed wood. The VP is a high-redox-potential enzyme and could be used to detoxify reactive oxygen species (ROS), which accumulate in plants during biotic and abiotic stresses. We cloned the VP gene and expressed it via the Agrobacterium transformation procedure in transgenic tobacco plants to assay their tolerance to different abiotic stress conditions. Thirty independent T2 transgenic VP lines overexpressing the fungal Bjerkandera adustaVP gene were selected on kanamycin. The VP22, VP24, and VP27 lines showed significant manganese peroxidase (MnP) activity. The highest was VP22, which showed 10.87-fold more manganese peroxidase activity than the wild-type plants and led to a 34% increase in plant height and 28% more biomass. The VP22, VP24, and VP27 lines showed enhanced tolerance to drought, 200 mM NaCl, and 400 mM sorbitol. Also, these transgenics displayed significant tolerance to methyl viologen, an active oxygen-generating compound. The present data indicate that overproducing the VP gene in plants increases significantly their biomass and the abiotic stress tolerance. The VP enzyme is an effective biotechnological tool to protect organisms against ROS. In transgenic tobacco plants, it improves drought, salt, and oxidative stress tolerance. Thus, the VP gene represents a great potential for obtaining stress-tolerant crops.

Expression of a spider venom peptide in transgenic tobacco confers insect resistance.

Spider venom contains a mixture of peptide toxins, some able to kill insects specifically to those considered as important pest. In this study, a peptide toxin produced by the Macrothele gigas spider, Magi 6, was cloned and expressed in tobacco plants, as this toxin has been shown to constitute an effective insecticide. For this purpose, a genetic construction for the cDNA that codifies for Magi 6 was subcloned in a plant expression vector using the 35S promoter and the 5'-end leader from tobacco mosaic virus, in order to transform tobacco leaf disks. The resulting plants demonstrated the presence of Magi 6 gene in the tobacco genome using PCR, and transcription of the cDNA was verified by means of RT-PCR. The expression of the Magi 6 peptide in tobacco was demonstrated by Western blot, which exhibited the expected size, thus suggesting a correct processing of the signal peptide. No morphological alterations in the different transgenic lines were observed, nor any change in plant growth. Subsequently, experiments were carried out challenging detached leaves or whole plants with the herbivorous insect Spodoptera frugiperda. The bioassays indicated that the transgenic lines were significantly more resistant than the wild type plants. This work demonstrated that the expression of Magi 6 peptide in transgenic plants conferred resistance to insect attack and opens the possibility of employing this peptide to improve the resistance of diverse plants.

The LEA proteins and trehalose loving couple: a step forward in anhydrobiotic engineering.

Adaptation to desiccation tolerance or anhydrobiosis has puzzled scientists for more than 300 years. Over the last few decades, considerable emphasis has been placed on understanding the role of two key molecules involved in anhydrobiosis: a peculiar disaccharide named trehalose and the hydrophilic LEA (Late Embryogenesis Abundant) proteins. In an article published in the Biochemical Journal in 2005, Alan Tunnacliffe and colleagues found that LEA proteins (alone, or more so in combination with trehalose) can protect stress-sensitive enzymes, such as citrate synthase and lactate dehydrogenase, from aggregation due to desiccation and freezing. Upon heat-stress, however, LEA proteins alone cannot prevent these enzymes from aggregating unless trehalose is present. This is the first report that LEA proteins can act as 'molecular shields' to prevent aggregation-induced cell damage due to water loss.

Trehalose metabolism: from osmoprotection to signaling.

Trehalose is a non-reducing disaccharide formed by two glucose molecules. It is widely distributed in Nature and has been isolated from certain species of bacteria, fungi, invertebrates and plants, which are capable of surviving in a dehydrated state for months or years and subsequently being revived after a few hours of being in contact with water. This disaccharide has many biotechnological applications, as its physicochemical properties allow it to be used to preserve foods, enzymes, vaccines, cells etc., in a dehydrated state at room temperature. One of the most striking findings a decade ago was the discovery of the genes involved in trehalose biosynthesis, present in a great number of organisms that do not accumulate trehalose to significant levels. In plants, this disaccharide has diverse functions and plays an essential role in various stages of development, for example in the formation of the embryo and in flowering. Trehalose also appears to be involved in the regulation of carbon metabolism and photosynthesis. Recently it has been discovered that this sugar plays an important role in plant-microorganism interactions.

GWAS to Identify Genetic Loci for Resistance to Yellow Rust in Wheat Pre-Breeding Lines Derived From Diverse Exotic Crosses.

Yellow rust (YR) or stripe rust, caused by Puccinia striformis f. sp tritici Eriks (Pst), is a major challenge to resistance breeding in wheat. A genome wide association study (GWAS) was performed using 22,415 single nucleotide polymorphism (SNP) markers and 591 haplotypes to identify genomic regions associated with resistance to YR in a subset panel of 419 pre-breeding lines (PBLs) developed at International Center for Maize and Wheat Improvement (CIMMYT). The 419 PBLs were derived from an initial set of 984 PBLs generated by a three-way crossing scheme (exotic/elite1//elite2) among 25 best elites and 244 exotics (synthetics, landraces) from CIMMYT's germplasm bank. For the study, 419 PBLs were characterized with 22,415 high-quality DArTseq-SNPs and phenotyped for severity of YR disease at five locations in Mexico. A population structure was evident in the panel with three distinct subpopulations, and a genome-wide linkage disequilibrium (LD) decay of 2.5 cM was obtained. Across all five locations, 14 SNPs and 7 haplotype blocks were significantly (P < 0.001) associated with the disease severity explaining 6.0 to 14.1% and 7.9 to 19.9% of variation, respectively. Based on average LD decay of 2.5 cM, identified 14 SNP-trait associations were delimited to seven quantitative trait loci in total. Seven SNPs were part of the two haplotype blocks on chromosome 2A identified in haplotypes-based GWAS. In silico analysis of the identified SNPs showed hits with interesting candidate genes, which are related to pathogenic process or known to regulate induction of genes related to pathogenesis such as those coding for glunolactone oxidase, quinate O-hydroxycinnamoyl transferase, or two-component histidine kinase. The two-component histidine kinase, for example, acts as a sensor in the perception of phytohormones ethylene and cytokinin. Ethylene plays a very important role in regulation of multiple metabolic processes of plants, including induction of defense mechanisms mediated by jasmonate. The SNPs linked to the promising genes identified in the study can be used for marker-assisted selection.

Improvement of drought tolerance and grain yield in common bean by overexpressing trehalose-6-phosphate synthase in rhizobia.

Improving stress tolerance and yield in crops are major goals for agriculture. Here, we show a new strategy to increase drought tolerance and yield in legumes by overexpressing trehalose-6-phosphate synthase in the symbiotic bacterium Rhizobium etli. Phaseolus vulgaris (common beans) plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene had more nodules with increased nitrogenase activity and higher biomass compared with plants inoculated with wild-type R. etli. In contrast, plants inoculated with an R. etli mutant in trehalose-6-phosphate synthase gene had fewer nodules and less nitrogenase activity and biomass. Three-week-old plants subjected to drought stress fully recovered whereas plants inoculated with a wild-type or mutant strain wilted and died. The yield of bean plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene and grown with constant irrigation increased more than 50%. Macroarray analysis of 7,200 expressed sequence tags from nodules of plants inoculated with the strain overexpressing trehalose-6-phosphate synthase gene revealed upregulation of genes involved in stress tolerance and carbon and nitrogen metabolism, suggesting a signaling mechanism for trehalose. Thus, trehalose metabolism in rhizobia is key for signaling plant growth, yield, and adaptation to abiotic stress, and its manipulation has a major agronomical impact on leguminous plants.

Insights on the evolution of trehalose biosynthesis.

BACKGROUND: The compatible solute trehalose is a non-reducing disaccharide, which accumulates upon heat, cold or osmotic stress. It was commonly accepted that trehalose is only present in extremophiles or cryptobiotic organisms. However, in recent years it has been shown that although higher plants do not accumulate trehalose at significant levels they have actively transcribed genes encoding the corresponding biosynthetic enzymes. RESULTS: In this study we show that trehalose biosynthesis ability is present in eubacteria, archaea, plants, fungi and animals. In bacteria there are five different biosynthetic routes, whereas in fungi, plants and animals there is only one. We present phylogenetic analyses of the trehalose-6-phosphate synthase (TPS) and trehalose-phosphatase (TPP) domains and show that there is a close evolutionary relationship between these domains in proteins from diverse organisms. In bacteria TPS and TPP genes are clustered, whereas in eukaryotes these domains are fused in a single protein. CONCLUSION: We have demonstrated that trehalose biosynthesis pathways are widely distributed in nature. Interestingly, several eubacterial species have multiple pathways, while eukaryotes have only the TPS/TPP pathway. Vertebrates lack trehalose biosynthetic capacity but can catabolise it. TPS and TPP domains have evolved mainly in parallel and it is likely that they have experienced several instances of gene duplication and lateral gene transfer.

Trehalose-6-phosphate synthase as an intrinsic selection marker for plant transformation.

Insertion of foreign DNA into plant genomes occurs randomly and with low frequency. Hence, a selectable marker is generally required to identify transgenic plants. Until now, all selection systems have been based on the use of non-plant genes, derived from microorganisms and usually conferring antibiotic or herbicide resistance. The use of microorganism-derived genes however has raised biosafety concerns. We have developed a novel selection system based on enhancing the expression of a plant-intrinsic gene and the use of a harmless selection agent. Selection takes advantage of the reduced glucose sensitivity of seedlings with enhanced expression of AtTPS1, a gene encoding trehalose-6-P synthase. As a result, transformants can be identified as developing green seedlings amongst the background of small, pale non-transformed plantlets on high glucose medium. In addition, vegetative regeneration of tobacco leaf explants is very sensitive to high external glucose. Overexpression of AtTPS1 in tobacco allows selecting glucose insensitive transgenic shoots.

Phylogenetic and biochemical characterisation of a recombinant laccase from Trametes versicolor.

Laccases are important enzymes for bioremediation and the best-characterised are from the fungus Trametes versicolor. Here, we describe the cloning and characterisation of a new variant of laccase from T. versicolor and its expression in Saccharomyces cerevisiae. We have performed a sequence-based analysis of Trametes laccases that leads to a proposal for a new nomenclature of this fungus laccases according to their phylogenetic relationships since their nomenclature based on IPs is ambiguous. We also describe the kinetic properties of the recombinant enzyme.

Draft Genome Sequence of Arthrobacter chlorophenolicus Strain Mor30.16, Isolated from the Bean Rhizosphere.

Bacteria of the genus Arthrobacter are commonly found in the soil and plant rhizosphere. In this study we report the draft genome of Arthrobacter chlorophenolicus strain Mor30.16 that was isolated from rhizosphere of beans grown in Cuernavaca Morelos, Mexico. This strain promotes growth and ameliorates drought stress in bean plants.

New selection marker for plant transformation.

A number of systems to insert foreign DNA into a plant genome have been developed so far. However, only a small percentage of transgenic plants are obtained using any of these methods. Stable transgenic plants are selected by co-introduction of a selectable marker gene, which in most cases are genes that confer resistance against antibiotics or herbicides. In this chapter we describe a new method for selection of transgenic plants after transformation. The selection agent used is the nontoxic and common sugar glucose. Wild-type Arabidopsis thaliana plantlets that have been germinated on glucose have small white cotyledons and remain petite because the external sugar switches off the photosynthetic mechanism. The selectable marker gene encodes the essential trehalose-6-phophate synthase, AtTPS1, that catalyzes the first reaction of the two-step trehalose synthesis. Upon ectopic expression of AtTPS1 driven by the 35S promoter, transformed Arabidopsis thaliana plants became insensitive to glucose in comparison to wild-type plants. After transformation using AtTPS1 as a selection marker and 6% glucose as selection agent it is possible to single out the green and normal sized transgenic plants amid the nontransformed plantlets.

Microorganisms associated to tomato seedlings growing in saline culture act as osmoprotectant.

Less than 0.5% of total water in the world is available for human consumption and agriculture. The major part of the world's water is saline and salinity in soils interferes in germination of seeds and the posterior development of the plant. In order to increase the osmotolerance of tomato, seedlings were associated with Azospirillum brasilense Cd, Azospirillum brasilense Cd transformed bacteria with a plasmid harboring a trehalose biosynthesis gene-fusion or Chlorella vulgaris. Two plant culture media: Hydroponic and Murashige and Skoog were tested. In the first set of studies seedlings were associated to single free cells meanwhile in a second set single and combined free cells were studied. A positive interaction between transformed Azospirillum and Chlorella vulagris and tomato plants was observed. Seedlings showed a salt concentration tolerance, as sodium chloride, up to 200 mM. According to our results, the association of plants with A. brasilense Cd-BIF and C. vulgaris is a viable approach to increase their salt tolerance and biomass, as consequence the possible use of sea water to irrigate horticultural plants.

Trehalose accumulation in Azospirillum brasilense improves drought tolerance and biomass in maize plants.

Bacteria of the genus Azospirillum increase the grain yield of several grass crops. In this work the effect of inoculating maize plants with genetically engineered Azospirillum brasilense for trehalose biosynthesis was determined. Transformed bacteria with a plasmid harboring a trehalose biosynthesis gene-fusion from Saccharomyces cerevisiae were able to grow up to 0.5 M NaCl and to accumulate trehalose, whereas wild-type A. brasilense did not tolerate osmotic stress or accumulate significant levels of the disaccharide. Moreover, 85% of maize plants inoculated with transformed A. brasilense survived drought stress, in contrast with only 55% of plants inoculated with the wild-type strain. A 73% increase in biomass of maize plants inoculated with transformed A. brasilense compared with inoculation with the wild-type strain was found. In addition, there was a significant increase of leaf and root length in maize plants inoculated with transformed A. brasilense. Therefore, inoculation of maize plants with A. brasilense containing higher levels of trehalose confers drought tolerance and a significant increase in leaf and root biomass. This work opens the possibility that A. brasilense modified with a chimeric trehalose biosynthetic gene from yeast could increase the biomass, grain yield and stress tolerance in other relevant crops.

Microorganisms associated to tomato seedlings growing in saline culture act as osmoprotectant

Less than 0.5% of total water in the world is available for human consumption and agriculture. The major part of the world's water is saline and salinity in soils interferes in germination of seeds and the posterior development of the plant. In order to increase the osmotolerance of tomato, seedlings were associated with Azospirillum brasilense Cd, Azospirillum brasilense Cd transformed bacteria with a plasmid harboring a trehalose biosynthesis gene-fusion or Chlorella vulgaris. Two plant culture media: Hydroponic and Murashige and Skoog were tested. In the first set of studies seedlings were associated to single free cells meanwhile in a second set single and combined free cells were studied. A positive interaction between transformed Azospirillum and Chlorella vulagris and tomato plants was observed. Seedlings showed a salt concentration tolerance, as sodium chloride, up to 200 mM. According to our results, the association of plants with A. brasilense Cd-BIF and C. vulgaris is a viable approach to increase their salt tolerance and biomass, as consequence the possible use of sea water to irrigate horticultural plants.

A bifunctional TPS-TPP enzyme from yeast confers tolerance to multiple and extreme abiotic-stress conditions in transgenic Arabidopsis.

Improving stress tolerance is a major goal for agriculture. Trehalose is a key molecule involved in drought tolerance in anhydrobiotic organisms. Here we describe the construction of a chimeric translational fusion of yeast trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase. This construct was overexpressed in yeast cells displaying both TPS and TPP enzyme activities and trehalose biosynthesis capacity. In Arabidopsis thaliana, the gene fusion was overexpressed using either the 35S promoter or the stress-regulated rd29A promoter. Transgene insertion in the genome was checked by PCR and transcript expression by RT-PCR. Several independent homozygous lines were selected in the presence of kanamycin and further analyzed. Trehalose was accumulated in all these lines at low levels. No morphological or growth alterations were observed in lines overexpressing the TPS1-TPS2 construct, whereas plants overexpressing the TPS1 alone under the control of the 35S promoter had aberrant growth, color and shape. TPS1-TPS2 overexpressor lines were glucose insensitive, consistent with a suggested role of trehalose/T6P in modulating sugar sensing and carbohydrate metabolism. Moreover, TPS1-TPS2 lines displayed a significant increase in drought, freezing, salt and heat tolerance. This is the first time that trehalose accumulation in plants is shown to protect against freezing and heat stress. Therefore, these results demonstrate that engineering trehalose metabolism with a yeast TPS-TPP bifunctional enzyme confers multiple stress protection in plants, comprising a potential tool to improve stress-tolerance in crops.

Betaine aldehyde dehydrogenase from Pseudomonas aeruginosa: cloning, over-expression in Escherichia coli, and regulation by choline and salt.

In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (BADH) may play a dual role assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacteria against the high-osmolarity stress prevalent in the infected tissues. We cloned the P. aeruginosa BADH gene and expressed the BADH protein in Escherichia coli. The recombinant protein appears identical to its native counterpart, as judged by Western blot, N-terminal amino acid sequence, tryptophan-fluorescence emission spectra, circular-dichroism spectroscopy, size-exclusion chromatography, and kinetic properties. Computational analysis indicated that the promoter sequence of the putative operon that includes the BADH gene has a consensus-binding site for the choline-sensing transcription repressor BetI, and putative boxes for ArcA and Lrp transcription factors but no known elements of response to osmotic stress. This is consistent with the strong induction of BADH expression by choline and with the lack of effect of NaCl. As there were significant amounts of BADH protein and activity in P. aeruginosa cells grown on glucose plus choline, as well as the BADH activity exhibiting tolerance to salt, it is likely that glycine betaine is synthesized in vivo and could play an important osmoprotectant role under conditions of infection.

Stress tolerance and glucose insensitive phenotypes in Arabidopsis overexpressing the CpMYB10 transcription factor gene.

The resurrection plant Craterostigma plantagineum has the ability to survive complete dehydration. In an attempt to further understand desiccation tolerance in this plant, the CpMYB10 transcription factor gene was functionally characterized. CpMYB10 is rapidly induced by dehydration and abscisic acid (ABA) treatments in leaves and roots, but no expression was detected in fully hydrated tissues. Electrophoretic mobility shift assay experiments showed binding of rCpMYB10 to specific mybRE elements within the LEA Cp11-24 and CpMYB10 promoters. Localization of CpMYB10 transcript by in situ reverse transcription-PCR reactions showed expression in vascular tissues, parenchyma, and epidermis both in leaves and roots in response to ABA. Transgenic Arabidopsis plants transformed with CpMYB10 promoter fused to GUS gene showed reporter expression under ABA and stress conditions in several organs. Overexpression of CpMYB10 cDNA in Arabidopsis led to desiccation and salt tolerance of transgenics lines. Interestingly, it was found that plants overexpressing CpMYB10 exhibited Glc-insensitive and ABA hypersensitive phenotypes. Therefore, our results indicate that CpMYB10 in Arabidopsis is mediating stress tolerance and altering ABA and Glc signaling responses.