Engineering Stress Tolerance Research Articles

Pathogenesis-Related Gene, JcPR-10a from Jatropha curcas Exhibit RNase and Antifungal Activity

The pathogenesis-related proteins have a broad spectrum of roles, ranging from seed germination, development to resistance. The PR-10 is a multigene family differing from other PR proteins in being intracellular, small and acidic with similar 3D structures. We have isolated JcPR-10a cDNA with an ORF of 483bp from J. curcas, an important biofuel crop grown in the wastelands of India. JcPR-10a gets clustered with dicots in phylogenetic tree. The genomic organisation analysis of JcPR-10a revealed the presence of an intron at conserved 185bp position. Transcript expression of JcPR-10a was upregulated in response to different stimuli such as NaCl, salicylic acid, methyl jasmonate and M. phaseolina. In response to SA and Macrophomina the transcript was found increased at 48h, however, in case of NaCl and MeJa a strong induction was observed at 12h which decreased at 48h. We first time report the transcript up regulation of PR-10 gene by Macrophomina, a pathogen causing collar rot in Jatropha. The recombinant E. coli cells showed better growth in LB medium supplemented with NaCl, whereas growth of recombinant cells was inhibited in LB medium supplemented with KCl, mannitol, sorbitol, methyl jasmonate and salicylic acid. The JcPR-10a protein was overexpressed in E. coli cells, and was purified to homogeneity, the purified protein exhibited RNase and DNase activity. Furthermore, the protein also showed antifungal activity against Macrophomina, indicating that JcPR-10a can serve as an important candidate to engineer stress tolerance in Jatropha as well as other plants susceptible to collar rot by Macrophomina.

Differential contribution of individual dehydrin genes from Physcomitrella patens to salt and osmotic stress tolerance

The moss Physcomitrella patens can withstand extreme environmental conditions including drought and salt stress. Tolerance to dehydration in mosses is thought to rely on efficient limitation of stress-induced cell damage and repair of cell injury upon stress relief. Dehydrin proteins (DHNs) are part of a conserved cell protecting mechanism in plants although their role in stress tolerance is not well understood. Four DHNs and two DHN-like proteins were identified in the predicted proteome of P. patens. Expression of PpDHNA and PpDHNB was induced by salt and osmotic stress and controlled by abscisic acid. Subcellular localization of the encoded proteins suggested that these dehydrins are localized in cytosol and accumulate near membranes during stress. Comparative analysis of dhnA and dhnB targeted knockout mutants of P. patens revealed that both genes play a role in cellular protection during salt and osmotic stress, although PpDHNA has a higher contribution to stress tolerance. Overexpression of PpDHNA and PpDHNB genes in transgenic Arabidopsis improved rosette and root growth in stress conditions, although PpDHNA was more efficient in this role. These results suggest that specific DHNs contribute considerably to the high stress tolerance of mosses and offer novel tools for genetic engineering stress tolerance of higher plants.

Deciphering energy‐associated gene networks operating in the response of Arabidopsis plants to stress and nutritional cues

Plants need to continuously adjust their transcriptome in response to various stresses that lead to inhibition of photosynthesis and the deprivation of cellular energy. This adjustment is triggered in part by a coordinated re-programming of the energy-associated transcriptome to slow down photosynthesis and activate other energy-promoting gene networks. Therefore, understanding the stress-related transcriptional networks of genes belonging to energy-associated pathways is of major importance for engineering stress tolerance. In a bioinformatics approach developed by our group, termed 'gene coordination', we previously divided genes encoding for enzymes and transcription factors in Arabidopsis thaliana into three clusters, displaying altered coordinated transcriptional behaviors in response to multiple biotic and abiotic stresses (Plant Cell, 23, 2011, 1264). Enrichment analysis indicated further that genes controlling energy-associated metabolism operate as a compound network in response to stress. In the present paper, we describe in detail the network association of genes belonging to six central energy-associated pathways in each of these three clusters described in our previous paper. Our results expose extensive stress-associated intra- and inter-pathway interactions between genes from these pathways, indicating that genes encoding proteins involved in energy-associated metabolism are expressed in a highly coordinated manner. We also provide examples showing that this approach can be further utilized to elucidate candidate genes for stress tolerance and functions of isozymes.

Role of DREBs in regulation of abiotic stress responses in plants

Abiotic stresses such as drought, high salinity, and cold are common adverse environmental conditions that significantly influence plant growth and productivity worldwide. The phytohormone abscisic acid (ABA) plays an important role in physiological and developmental responses as well as in co-ordinating various stress signal transduction pathways in plants. DREBs (dehydration responsive element binding) are important plant transcription factors (TFs) that regulate the expression of many stress-inducible genes mostly in an ABA-independent manner and play a critical role in improving the abiotic stress tolerance of plants by interacting with a DRE/CRT cis-element present in the promoter region of various abiotic stress-responsive genes. This review summarizes recent studies highlighting the role of the DRE-binding family of TFs in the adaptive responses to different abiotic stresses and their structural and functional characters with emphasis on the expression and regulation of DREBs. The practical and application value of DREBs in crop improvement, such as stress tolerance engineering as well as marker-assisted selection (MAS), has also been discussed.

The soybean GmbZIP1 transcription factor enhances multiple abiotic stress tolerances in transgenic plants

Abscisic acid (ABA)-responsive element binding proteins (AREBs) are basic domain/leucine zipper transcription factors that bind to the ABA-responsive element (ABRE) in the promoter regions of ABA-inducible genes in plants. A novel bZIP transcription factor gene, GmbZIP1, encoding 438 amino acids with a conserved bZIP domain composed of 60 amino acids was isolated from salt-tolerant soybean cv. Tiefeng 8. Southern blotting showed that only one copy was present in the soybean genome. Phylogenetic analyses showed that GmbZIP1 belonged to the AREB subfamily of the bZIP family and was most closely related to AtABF2 and OsTRAB1. The expression of GmbZIP1 was highly induced by ABA, drought, high salt and low temperature; and GmbZIP1 was expressed in soybean roots, stems and leaves under different stress conditions. GmbZIP1 was localized inside the nuclei of transformed onion epidermal cells. Overexpression of GmbZIP1 enhanced the responses of transgenic plants to ABA and triggered stomatal closure under stresses, potentially leading to improved tolerances to several abiotic stresses such as high salt, low temperature and drought in transgenic plants. Furthermore, overexpression of GmbZIP1 affected the expression of some ABA or stress-related genes involved in regulating stomatal closure in Arabidopsis under ABA, drought and high salt stress conditions. A few AREB elements were detected in the promoter region of those ABA or stress-related genes, suggesting that GmbZIP1 regulates the ABA response or stomatal closure mediated by those downstream genes in transgenic Arabidopsis. Moreover, GmbZIP1 was used to improve the drought tolerance trait of Chinese wheat varieties BS93. Functional analysis showed that overexpression of GmbZIP1 enhanced the drought tolerance of transgenic wheat, and transcripts of GmbZIP1 were detected in transgenic wheat using RT-PCR. In addition, GmbZIP1 overexpression did not result in growth retardation in all transgenic plants, suggesting that GmbZIP1 may be a valuable genetic resource for engineering stress tolerance of crops.

Transcription factors as tools to engineer enhanced drought stress tolerance in plants

Plant growth and productivity are greatly affected by abiotic stresses such as drought, salinity, and temperature. Drought stress is one of the major limitations to crop productivity worldwide due to its multigene nature, making the production of transgenic crops a challenging prospect. To develop crop plant with enhanced tolerance of drought stress, a basic understanding of physiological, biochemical, and gene regulatory networks is essential. In the signal transduction network that leads from the perception of stress signals to the expression of stress-responsive genes, transcription factors (TFs) play an essential role. Because TFs, as opposed to most structural genes, tend to control multiple pathways steps, they have emerged as powerful tools for the manipulation of complex metabolic pathways in plants. One such class of TFs is DREB/CBF that binds to drought responsive cis-acting elements. Transgenic plants have been developed with enhanced stress tolerance by manipulating the expression of DREB/CBF. Recently the functions of an increasing number of plant TFs are being elucidated and increased understanding of these factors in controlling drought stress response has lead to practical approaches for engineering stress tolerance in plants. The utility of the various TFs in plant stress research we review is illustrated by several published examples. The manipulation of native plant regularity networks therefore represents a new era for genetically modified crops. This review focuses on the recent understanding, latest advancements related to TFs and present status of their deployment in developing stress tolerant transgenic plants.

Transcriptome analysis reveals absence of unintended effects in drought-tolerant transgenic plants overexpressing the transcription factor ABF3

BackgroundPlants engineered for abiotic stress tolerance may soon be commercialized. The engineering of these plants typically involves the manipulation of complex multigene networks and may therefore have a greater potential to introduce pleiotropic effects than the simple monogenic traits that currently dominate the plant biotechnology market. While research on unintended effects in transgenic plant systems has been instrumental in demonstrating the substantial equivalence of many transgenic plant systems, it is essential that such analyses be extended to transgenic plants engineered for stress tolerance. Drought-tolerant Arabidopsis thaliana were engineered through overexpression of the transcription factor ABF3 in order to investigate unintended pleiotropic effects. In order to eliminate position effects, the Cre/lox recombination system was used to create control plant lines that contain identical T-DNA insertion sites but with the ABF3 transgene excised. This additionally allowed us to determine if Cre recombinase can cause unintended effects that impact the transcriptome.ResultsMicroarray analysis of control plant lines that underwent Cre-mediated excision of the ABF3 transgene revealed only two genes that were differentially expressed in more than one plant line, suggesting that the impact of Cre recombinase on the transcriptome was minimal. In the absence of drought stress, overexpression of ABF3 had no effect on the transcriptome, but following drought stress, differences were observed in the gene expression patterns of plants overexpressing ABF3 relative to control plants. Examination of the functional distribution of the differentially expressed genes revealed strong similarity indicating that unintended pathways were not activated.ConclusionsThe action of ABF3 is tightly controlled in Arabidopsis. In the absence of drought stress, ectopic activation of drought response pathways does not occur. In response to drought stress, overexpression of ABF3 results in a reprogramming of the drought response, which is characterized by changes in the timing or strength of expression of some drought response genes, without activating any unexpected gene networks. These results illustrate that important gene networks are highly regulated in Arabidopsis and that engineering stress tolerance may not necessarily cause extensive changes to the transcriptome.

Enhanced Tolerance to Multiple Abiotic Stresses in Transgenic Alfalfa Accumulating Trehalose

ABSTRACTAlfalfa (Medicago sativaL.) is an important forage crop in many countries with high biomass production and the third‐most‐cultivated crop in the United States, with high protein content and potential use as biofuel. Here we describe the use of a chimeric translational fusion of yeast trehalose‐6‐phosphate synthase (EC 2.4.1.15) and trehalose‐6‐phosphate phosphatase (EC 3.1.3.12) genes to engineer stress tolerance in alfalfa. The gene fusion was overexpressed using either the constitutive35Spromoter or the stress‐regulatedrd29Apromoter. Transgene insertion in the genome was checked by polymerase chain reaction and transcript expression by reverse transcription polymerase chain reaction. Several independent transgenic plants were selected in the presence of kanamycin and further analyzed. Yeast gene encoding trehalose‐6‐phosphate synthase (TPS1)–yeast gene encoding trehalose‐6‐phosphate phosphatase (TPS2) gene expression under the35Spromoter led to plants with stunted growth and less biomass, whereasTPS1‐TPS2expression driven by therd29Apromoter improved growth and provoked a significant increase in plant biomass at the foliage level in the different transgenic lines. Trehalose accumulated in all the different lines at similar levels under stress conditions. Transgenic plants displayed a significant increase in drought, freezing, salt, and heat tolerance. This is the first time that genetic engineering of trehalose metabolism is reported in alfalfa and demonstrates that it can improve multiple stress protection in this crop. Thus, the yeast TPS‐TPP fusion protein represents a great potential for generating stress‐tolerant crop plants for agriculture.

Physiological Roles of Polyols in Horticultural Crops

Sucrose is generally considered the primary photosynthate in plants; however, many horticultural crops, including rosaceous fruit trees, synthesize and use polyols (sugar alcohols). This review describes recent progress in physiological research on the metabolism and transport of sorbitol in rosaceous fruit trees, and of mannitol, another common polyol, in horticultural crops. Studies on various polyols other than sorbitol (in rosaceous fruit trees) and mannitol are then described. Polyols play a role not only in the translocation and storage of photosynthates but also in biotic and abiotic responses in many horticultural crops; therefore, this review also provides insights into the effects of polyol metabolism on the biochemical mechanisms of pathogenicity and environmental stress tolerance, including a novel strategy for engineering stress tolerance.

Trehalose Metabolites in Arabidopsis-elusive, active and central.

Trehalose is an alpha, alpha-1, 1-linked glucose disaccharide. In plants, trehalose is synthesized in two steps. Firstly, trehalose-6-phosphate synthase (TPS) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate (T6P); secondly, T6P-phosphatase (TPP) converts T6P into trehalose and Pi. Trehalose is further cleaved into glucose by trehalase. In extracts of most plants, including Arabidopsis, levels of both trehalose and T6P are low, nearing detection limits, and this has delayed research into their function. Trehalose is transported widely in plants, but transport of T6P is not thought to occur except possibly at the subcellular level. Feeding trehalose to Arabidopsis seedlings alters carbon allocation with massive starch accumulation in cotyledons and leaves and absence of starch and growth in shoot and root apices.The Arabidopsis genome has experienced extensive radiation of genes likely encoding enzymes of T6P metabolism: 4 and 10 genes are found with homology to TPS and TPP respectively and 7 genes are found with homology to both TPS and TPP. Complementation of Saccharomyces cerevisiae mutants has shown that AtTPS1, AtTPPA and AtTPPB are functional enzymes. In contrast just a single gene encoding a protein with trehalase activity has been found. Whilst most TPS proteins appear cytosolic, strikingly, some TPPs appear targeted to chloroplasts; trehalase on the other hand is extracellular. Transporters of trehalose and T6P have yet to be described. Arabidopsis tps1 mutants are embryo lethal and results suggest that T6P is essential for several other steps in development including root growth and floral transition. Accordingly, altering T6P content has a profound effect on plant habitus and impacts metabolite profiles, sugar utilization and photosynthesis. These large effects have hindered dissection of cause and effect. In contrast, plants with large alterations in sucrose-6-phosphate concentrations are indistinguishable from wild type, suggesting very different functions for these compounds. Recently, T6P at low micromolar concentrations has been shown in vitro and in vivo to inhibit SnRK1 of the SNF1/AMPK group of protein kinases. This supports a function for T6P as a sugar signaling molecule integrating metabolism and development in plants in relation to carbon supply.Genetic engineering of Arabidopsis as well as tobacco, potato and rice with TPS or TPS/TPP protein fusions reveals that trehalose metabolism also mediates multiple abiotic stress tolerances. Trehalose applications also mediate biotic stress resistances. Both Escherichia coli and Saccharomyces cerevisiae TPS/TPP protein fusions can be used to engineer stress tolerance suggesting that metabolites rather than proteins of the trehalose pathway are key stress tolerance elicitors. Results underscore the central role of trehalose metabolites in integrating carbon metabolism and stress responses with plant development.

OsLEA3, a late embryogenesis abundant protein gene from rice, confers tolerance to water deficit and salt stress to transgenic rice

OsLEA3 is a late embryogenesis abundant group 3 protein. The OsLEA3 gene located on chromosome 5 of rice (Oryza sativa L.) includes one intron and two exons and encodes a protein of 200 amino acid residues. Expression analysis revealed that OsLEA3 was induced by water deficit and salt stress. Overexpression of the OsLEA3 gene in the transgenic rice plants allowed us to test the role of the OsLEA3 protein in stress tolerance. The accumulation of the OsLEA3 protein in the vegetative tissues of transgenic rice plants enhanced their tolerance to water deficit and salt stress. These results demonstrate a role for the OsLEA3 protein in stress protection and suggest the potential of the OsLEA3 gene for genetic engineering of stress tolerance.

Engineering for Drought Tolerance in Horticultural and Ornamental Plants

Drought is one of the major factors limiting plant growth and productivity. Plant adaptation to drought is dependent on molecular networks for drought perception, signal transduction, expression of a subset of genes and production of metabolites that protect and maintain the structure of cellular components. In general, the drought response pathways can be classified into two categories: one is dependent on the stress hormone abscisic acid (ABA) and the other is ABA-independent. Many genes in these pathways have been identified, thereby providing guidance in choosing genes for engineering of drought tolerance. The review highlights the genes that mediate drought response and tolerance, and discusses lessons learned from engineering for drought tolerance in model plants, such as Arabidopsis, rice and tobacco. Because success of drought tolerance engineering is dependent on not only protein coding regions but also appropriate promoters, this article also reviews the promoters that are crucial for successful engineering of stress tolerance.

An engineered sorbitol cycle alters sugar composition, not growth, in transformed tobacco

Many efforts have been made to engineer stress tolerance by accumulating polyols. Transformants that accumulate polyols often show growth inhibition, because polyols are synthesized as a dead-end product in plants that do not naturally accumulate polyols. Here, we show a novel strategy in which a sorbitol cycle was engineered by introducing apple cDNA encoding NAD-dependent sorbitol dehydrogenase (SDH) in addition to sorbitol-6-phosphate dehydrogenase (S6PDH). Tobacco plants transformed only with S6PDH showed growth inhibition, and very few transformants were obtained. In contrast, many transgenic plants with both S6PDH and SDH were easily obtained, and their growth was normal despite their accumulation of sorbitol. Interestingly, the engineered sorbitol cycle enhanced the accumulation of sucrose instead of fructose that was expected to be increased. Sucrose, rather than fructose, was also increased in the immature fruit of tomato plants transformed with an antisense fructokinase gene in which the phosphorylation of fructose was inhibited. A common phenomenon was observed in the metabolic engineering of two different pathways, showing the presence of homeostatic regulation of fructose levels.

Role of DREB transcription factors in abiotic and biotic stress tolerance in plants

Abiotic and biotic stresses negatively influence survival, biomass production and crop yield. Being multigenic as well as a quantitative trait, it is a challenge to understand the molecular basis of abiotic stress tolerance and to manipulate it as compared to biotic stresses. Lately, some transcription factor(s) that regulate the expression of several genes related to stress have been discovered. One such class of the transcription factors is DREB/CBF that binds to drought responsive cis-acting elements. DREBs belong to ERF family of transcription factors consisting of two subclasses, i.e. DREB1/CBF and DREB2 that are induced by cold and dehydration, respectively. The DREBs are apparently involved in biotic stress signaling pathway. It has been possible to engineer stress tolerance in transgenic plants by manipulating the expression of DREBs. This opens an excellent opportunity to develop stress tolerant crops in future. This review intends to focus on the structure, role of DREBs in plant stress signaling and the present status of their deployment in developing stress tolerant transgenic plants.

Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance.

Abiotic stresses, such as drought, salinity, extreme temperatures, chemical toxicity and oxidative stress are serious threats to agriculture and the natural status of the environment. Increased salinization of arable land is expected to have devastating global effects, resulting in 30% land loss within the next 25 years, and up to 50% by the year 2050. Therefore, breeding for drought and salinity stress tolerance in crop plants (for food supply) and in forest trees (a central component of the global ecosystem) should be given high research priority in plant biotechnology programs. Molecular control mechanisms for abiotic stress tolerance are based on the activation and regulation of specific stress-related genes. These genes are involved in the whole sequence of stress responses, such as signaling, transcriptional control, protection of membranes and proteins, and free-radical and toxic-compound scavenging. Recently, research into the molecular mechanisms of stress responses has started to bear fruit and, in parallel, genetic modification of stress tolerance has also shown promising results that may ultimately apply to agriculturally and ecologically important plants. The present review summarizes the recent advances in elucidating stress-response mechanisms and their biotechnological applications. Emphasis is placed on transgenic plants that have been engineered based on different stress-response mechanisms. The review examines the following aspects: regulatory controls, metabolite engineering, ion transport, antioxidants and detoxification, late embryogenesis abundant (LEA) and heat-shock proteins.

THE ROLE OF MOLECULAR BIOLOGY IN ENHANCING THE SALT-TOLERANCE OF HORTICULTURAL CROPS

It has proved difficult to develop crops that are resistant to salinities beyond those normally experienced by the species from which that crop has evolved. Since advances in molecular biology do not appear to have helped greatly in the development of salt-tolerant genotypes, it is reasonable to reflect on the lack of progress. There is good evidence that salt tolerance in truly tolerant species (halophytes) is a multifaceted trait. In halophytes, tolerance depends upon compartmentation and compatible solutes, regulation of transpiration, control of ion leakage through the apoplast, membrane characteristics and the ability to tolerate low K:Na in the cytoplasm. A range of traits has also been demonstrated to be important in crops. This suggests that the overall trait, of salt tolerance is determined by a number of genes: that the trait is quantitative. Genetic markers can be identified for such quantitative traits. Recently, markers have been reported for traits associated with salt tolerance, but this has not so far helped with the identification of specific genes - although there is the potential for marker-assisted selection. Recent work on cold tolerance suggests that it may be possible to engineer regulatory genes: such genes may control a range of processes necessary for tolerance. If this proves possible for salt tolerance, then this may be an important advance in our ability to engineer stress tolerance into plants. Successful attempts have been made to transfer genes concerned with the synthesis of compatible solutes (osmolytes) into plants and claims made for enhanced tolerance. Whether tolerance is really enhanced is questionable as are the reasons for any putative enhancement.

Osmoregulation in the model organismEscherichia coli: genes governing the synthesis of glycine betaine and trehalose and their use in metabolic engineering of stress tolerance

Glycine betaine is known to be the preferred osmoprotectant in many bacteria, and glycine betaine accumulation has also been correlated with increased cold tolerance. Trehalose is often a minor osmoprotectant in bacteria and it is a major determinant for desiccation tolerance in many so-called anhydrobiotic organisms such as baker's yeast(Saccharomyces cerevisiae). Escherichia coli has two pathways for synthesis of these protective molecules; i.e., a two-step conversion of UDP-glucose and glucose-6-phosphate to trehalose and a two-step oxidation of externally-supplied choline to glycine betaine. The genes governing the choline-to-glycine betaine pathway have been studied inE. coli and several other bacteria and higher plants. The genes governing UDP-glucose-dependent trehalose synthesis have been studied inE. coli andS. cerevisiae. Because of their well-documented function in stress protection, glycine betaine and trehalose have been identified as targets for metabolic engineering of stress tolerance. Examples of this experimental approach include the expression of theE. coli betA andArthrobacter globiformis codA genes for glycine betaine synthesis in plants and distantly related bacteria, and the expression of theE. coli otsA and yeastTPS1 genes for trehalose synthesis in plants. The published data show that glycine betaine synthesis protects transgenic plants and phototrophic bacteria against stress caused by salt and cold. Trehalose synthesis has been reported to confer increased drought tolerance in transgenic plants, but it causes negative side effects which is of concern. Thus, the much-used model organismE. coli has now become a gene resource for metabolic engineering of stress tolerance.

Engineering Stress Tolerance in Maize

A wide range of environmental stresses (such as chilling, ozone, high light, drought, and heat) can damage crop plants, with consequent high annual yield losses. A common factor in all these unrelated adverse conditions, called oxidative stress, is the enhanced production of active oxygen species (AOS) within several subcellular compartments of the plant. AOS can react very rapidly with DNA, lipids and proteins, causing severe cellular damage. Under normal growth conditions, AOS are efficiently scavenged by both enzymatic and non-enzymatic detoxification mechanisms. Nevertheless, during prolonged stress conditions such detoxification systems get saturated and damage occurs. The main players within the defence system are superoxide dismutases, ascorbate peroxidase, and catalases. These enzymes directly eliminate the harmful AOS. By enhancing the levels of these proteins in transgenic plants via transformation technology the improvement of tolerance against oxidative stress is being attempted. In our research, we are generating transgenic maize lines that overproduce various antioxidative stress enzymes and we are assessing the performance of these plants during chilling stress.

Engineering stress tolerance in transgenic plants

Research in our laboratory has focused on the analysis of the functions of a variety of enzymes that are involved in the scavenging of reactive oxygen intermediates (ROI) such as superoxide radicals (·O2−) and hydrogen peroxide (H2O2). Recent work has been on transgenic plants that over-express glutathione S-transferases (GST) that also have glutathione peroxidase activity. Transgenic tobacco plants that contain gene constructs that encode two different tobacco GST’s had elevated levels of both GST and GPX activity. Analysis of mature vegetative transgenic tobacco plants that over-express GST/GPX failed to show any increase in paraquat tolerance or protection from photooxidative stress. However, seeds of these GST/GPX-expressing tobacco lines are capable of more rapid germination and seedling growth at low temperatures and at elevated salt concentrations. Reduced levels of lipid peroxidation were noted in GST/GPX-expressing seedling compared to control seedlings under both stressful and non-stressful conditions. In addition, GST/GPX-expressing seedlings significantly accumulated more oxidized glutathione (GSSG) than control seedlings during stress. These characteristics clearly indicate that over-expression of GST/GPX in transgenic seedlings can have substantial effects on their stress tolerance. Furthermore, it appears that this effect is due primarily to the elevated levels of GPX activity.

Osmoprotective compounds in the Plumbaginaceae: a natural experiment in metabolic engineering of stress tolerance.

In common with other zwitterionic quarternary ammonium compounds (QACs), glycine betaine acts as an osmoprotectant in plants, bacteria, and animals, with its accumulation in the cytoplasm reducing adverse effects of salinity and drought. For this reason, the glycine betaine biosynthesis pathway has become a target for genetic engineering of stress tolerance in crop plants. Besides glycine betaine, several other QAC osmoprotectants have been reported to accumulate among flowering plants, although little is known about their distribution, evolution, or adaptive value. We show here that various taxa of the highly stress-tolerant family Plumbaginaceae have evolved four QACs, which supplement or replace glycine betaine-namely, choline O-sulfate and the betaines of beta-alanine, proline, and hydroxyproline. Evidence from bacterial bioassays demonstrates that these QACs function no better than glycine betaine as osmoprotectants. However, the distribution of QACs among diverse members of the Plumbaginaceae adapted to different types of habitat indicates that different QACs could have selective advantages in particular stress environments. Specifically, choline O-sulfate can function in sulfate detoxification as well as in osmoprotection, beta-alanine betaine may be superior to glycine betaine in hypoxic saline conditions, and proline-derived betaines may be beneficial in chronically dry environments. We conclude that the evolution of osmoprotectant diversity within the Plumbaginaceae suggests additional possibilities to explore in the metabolic engineering of stress tolerance in crops.