Kandelia candel Thioredoxin f Confers Osmotic Stress Tolerance in Transgenic Tobacco
Xiaoshu JingJun YaoXujun MaYanli ZhangYuanling SunMin XiangPeichen HouNi-Ya LiRui ZhaoJinke LiXiaoyang ZhouShaoliang Chen
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Water deficit caused by osmotic stress and drought limits crop yield and tree growth worldwide. Screening and identifying candidate genes from stress-resistant species are a genetic engineering strategy to increase drought resistance. In this study, an increased concentration of mannitol resulted in elevated expression of thioredoxin f (KcTrxf) in the nonsecretor mangrove species Kandelia candel. By means of amino acid sequence and phylogenetic analysis, the mangrove Trx was classified as an f-type thioredoxin. Subcellular localization showed that KcTrxf localizes to chloroplasts. Enzymatic activity characterization revealed that KcTrxf recombinant protein possesses the disulfide reductase function. KcTrxf overexpression contributes to osmotic and drought tolerance in tobacco in terms of fresh weight, root length, malondialdehyde (MDA) content, and hydrogen peroxide (H2O2) production. KcTrxf was shown to reduce the stomatal aperture by enhancing K+ efflux in guard cells, which increased the water-retaining capacity in leaves under drought conditions. Notably, the abscisic acid (ABA) sensitivity was increased in KcTrxf-transgenic tobacco, which benefits plants exposed to drought by reducing water loss by promoting stomatal closure. KcTrxf-transgenic plants limited drought-induced H2O2 in leaves, which could reduce lipid peroxidation and retain the membrane integrity. Additionally, glutathione (GSH) contributing to reactive oxygen species (ROS) scavenging and transgenic plants are more efficient at regenerating GSH from oxidized glutathione (GSSG) under conditions of drought stress. Notably, KcTrxf-transgenic plants had increased glucose and fructose contents under drought stress conditions, presumably resulting from KcTrxf-promoted starch degradation under water stress. We conclude that KcTrxf contributes to drought tolerance by increasing the water status, by enhancing osmotic adjustment, and by maintaining ROS homeostasis in transgene plants.Keywords:
Kandelia candel
Osmotic shock
Glutathione reductase
Drought Tolerance
Malondialdehyde
Choline (Cho) is an essential nutrient for humans as well as the precursor of glycine betaine (GlyBet), an important compatible solute in eukaryotes that protects cells from osmotic stress caused by dehydrating conditions. The key enzyme for plant Cho synthesis is phosphoethanolamine N-methyltransferase (PEAMT), which catalyzes all three methylation steps, including the rate-limiting N-methylation of phosphoethanolamine. Herein, we report that the beneficial soil bacterium Bacillus subtilis (strain GB03) enhances Arabidopsis Cho and GlyBet synthesis associated with enhanced plant tolerance to osmotic stress. When stressed with 100 mM exogenous mannitol, GB03-exposed plants exhibit increased transcript level of PEAMT compared with stressed plants without bacterial exposure. Endogenous Cho and GlyBet metabolite pools were elevated by more than two- and fivefold, respectively, by GB03 treatment, consistent with increased stress tolerance. Moreover, in the xipotl mutant line with reduced Cho production, a loss of GB03-induced drought tolerance is observed. Osmotic-stressed plants with or without GB03 exposure show similar levels of abscsisic acid (ABA) accumulation in both shoots and roots, suggesting that GB03-induced osmoprotection is ABA independent. GB03 treatment also improves drought tolerance in soil-grown plants as characterized by phenotypic comparisons, supported by an elevated accumulation of osmoprotectants. These results provide a biological strategy to enhance Cho biosynthesis in plants and, in turn, increase plant tolerance to osmotic stress by elevating osmoprotectant accumulation.
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Escherichia coli cells growing under osmotic stress activate systems for the transport or synthesis of several organic osmolytes. Glutamate and trehalose syntheses seem to represent mechanisms for achieving a low level of osmotic tolerance. The uptake and synthesis of betaines represent mechanisms of achieving a high level of osmotic tolerance. The osmotically-controlled ProP and ProU systems are involved in the uptake of glycine betaine and proline. However, glycine betaine synthesis occurs only in the presence of the precursor molecule choline. The osmotically controlled genes governing the high-affinity uptake of choline are located in close proximity to those encoding the dehydrogenases involved in the oxidation of choline to glycine betaine, but represent a different transcriptional unit. It is not known if each of these systems has its own osmotic sensor or whether a common osmotic sensor regulates all cell osmolytes.
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The disulfide reducing enzymes glutathione reductase and thioredoxin reductase are highly conserved among bacteria, fungi, worms, and mammals. These proteins maintain intracellular redox homeostasis to protect the organism from oxidative damage. Here we demonstrate the absence of glutathione reductase in Drosophila melanogaster, identify a new type of thioredoxin reductase, and provide evidence that a thioredoxin system supports GSSG reduction. Our data suggest that antioxidant defense in Drosophila, and probably in related insects, differs fundamentally from that in other organisms.
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The mechanism of osmoadaptation in a salt-tolerant (1.2 M NaCl) bacterial isolate identified as Pseudomonas mendocina (N. J. Palleroni, M. Doudoroff, R. Y. Stanier, R. E. Solanes, and R. Mandel, J. Gen. Microbiol. 60:215-231, 1970) was investigated. In response to osmotic stress, this species accumulated a number of compatible solutes, the intracellular levels of which depended on both the osmolarity and the ionic composition of the growth medium. Glucosylglycerol [alpha-D-glucopyranosyl-alpha-(1-->2)-glycerol], N-acetylglutaminylglutamine amide, and L-alpha-glutamate were the major compatible solutes accumulated via de novo biosynthesis. Trehalose was also accumulated, but only in cells grown in the presence of high concentrations of sulfate or phosphate ions. Glycine betaine was accumulated only when supplied exogenously to cells grown at high osmolarity, and its accumulation caused a significant depletion of the intracellular pools of glucosylglycerol and glutamate. Glucosylglycerol was also found to accumulate in the type strains of P. mendocina and P. pseudoalcaligenes. This is the first report demonstrating the pivotal role of glucosylglycerol in osmoadaptation in a nonphotosynthetic microorganism.
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Glycinebetaine is an important non-toxic osmoprotectant, which is accumulated in higher plants under various stresses. The biosynthesis of glycinebetaine achieved via is a two-step oxidation from choline and betaine aldehyde, catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. Up-regulated gene expression of BADH and CMO induced by stress is clearly observed, but the signal transduction is poorly understood. Here, glycinebetaine accumulation in response to osmotic stress and growth recovery induced by exogenous glycinebetaine were observed in a watermelon cell line. When tracing back to the genome sequence of watermelon, it shows that there exists only one member of ClCMO or ClBADH corresponding to glycinebetaine biosynthesis. Both genes harbor a CGTCA-motif in their promoter region which is involved in methyl jasmonate (MeJA)-responsiveness. Amongst MeJA, Ethephon, abscisic acid (ABA), and salicylic acid (SA), MeJA was most effective in gene inducing the expression of ClCMO and ClBADH, and the accumulation of glycinebetaine could also reach an amount comparable to that after osmotic stress by mannitol. Moreover, when ibuprofen (IBU), a JA biosynthesis inhibitor, was pre-perfused into the cells before osmotic stress, glycinebetaine accumulation was suppressed significantly. Interestingly, newly grown cells can keep a high content of glycinebetaine when they are sub-cultured from osmotic stressed cells. This study suggests that osmotic stress induced glycinebetaine biosynthesis occurs via JA signal transduction and not only plays a key role in osmotic stress resistance but also contributes to osmotic stress hardening.
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Summary Bacillus subtilis , in its natural habitat, is regularly exposed to rapid changes in the osmolarity of its surrounding. As its primary survival strategy, it accumulates large amounts of the compatible solute proline by activating the de novo proline biosynthesis pathway and exploiting the glutamate pools. This osmotically‐induced biosynthesis requires activation of a SigA‐type promoter that drives the expression of the proHJ operon. Population‐wide studies have shown that the activity of the proHJ promoter correlates with the increased osmotic pressure of the environment. Therefore, the activation of the proHJ transcription should be an adequate measure of the adaptation to osmotic stress through proline synthesis in the absence of other osmoprotectants. In this study, we investigate the kinetics of the proHJ promoter activation and the early adaptation to mild osmotic upshift at the single‐cell level. Under these conditions, we observed a switching point and heterogeneous proline biosynthesis gene expression, where the subpopulation of cells showing active proHJ transcription is able to continuously divide, and those unresponsive to osmotic stress remain dormant. Additionally, we demonstrate that bactericidal antibiotics significantly upregulate proHJ transcription in the absence of externally imposed osmotic pressure, suggesting that the osmotically‐controlled proline biosynthesis pathway is also involved in the antibiotic‐mediated stress response.
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Genome-Wide Transcriptional Responses ofEscherichia coliK-12 to Continuous Osmotic and Heat Stresses
ABSTRACT Osmotic stress is known to increase the thermotolerance and oxidative-stress resistance of bacteria by a mechanism that is not adequately understood. We probed the cross-regulation of continuous osmotic and heat stress responses by characterizing the effects of external osmolarity (0.3 M versus 0.0 M NaCl) and temperature (43°C versus 30°C) on the transcriptome of Escherichia coli K-12. Our most important discovery was that a number of genes in the SoxRS and OxyR oxidative-stress regulons were up-regulated by high osmolarity, high temperature, or a combination of both stresses. This result can explain the previously noted cross-protection of osmotic stress against oxidative and heat stresses. Most of the genes shown in previous studies to be induced during the early phase of adaptation to hyperosmotic shock were found to be also overexpressed under continuous osmotic stress. However, there was a poorer overlap between the heat shock genes that are induced transiently after high temperature shifts and the genes that we found to be chronically up-regulated at 43°C. Supplementation of the high-osmolarity medium with the osmoprotectant glycine betaine, which reduces the cytoplasmic K + pool, did not lead to a universal reduction in the expression of osmotically induced genes. This finding does not support the hypothesis that K + is the central osmoregulatory signal in Enterobacteriaceae .
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