Heterologous Production of Glycine Betaine Using Synechocystis sp. PCC 6803-Based Chassis Lacking Native Compatible Solutes

Among compatible solutes, glycine betaine has various applications in the fields of nutrition, pharmaceuticals, and cosmetics. Currently, this compound can be extracted from sugar beet plants or obtained by chemical synthesis, resulting in low yields or high carbon footprint, respectively. Hence, in this work we aimed at exploring the production of glycine betaine using the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a photoautotrophic chassis. Synechocystis mutants lacking the native compatible solutes sucrose or/and glucosylglycerol—∆sps, ∆ggpS, and ∆sps∆ggpS—were generated and characterized. Under salt stress conditions, the growth was impaired and accumulation of glycogen decreased by ∼50% whereas the production of compatible solutes and extracellular polymeric substances (capsular and released ones) increased with salinity. These mutants were used as chassis for the implementation of a synthetic device based on the metabolic pathway described for the halophilic cyanobacterium Aphanothece halophytica for the production of the compatible solute glycine betaine. Transcription of ORFs comprising the device was shown to be stable and insulated from Synechocystis’ native regulatory network. Production of glycine betaine was achieved in all chassis tested, and was shown to increase with salinity. The introduction of the glycine betaine synthetic device into the ∆ggpS background improved its growth and enabled survival under 5% NaCl, which was not observed in the absence of the device. The maximum glycine betaine production [64.29 µmol/gDW (1.89 µmol/mg protein)] was reached in the ∆ggpS chassis grown under 3% NaCl. Taking into consideration this production under seawater-like salinity, and the identification of main key players involved in the carbon fluxes, this work paves the way for a feasible production of this, or other compatible solutes, using optimized Synechocystis chassis in a pilot-scale.

Major Compatible Solutes and Structural Adaptation of Proteins in Extremophiles

Extremophiles are the most ancient microbes on the Earth and also a center of attraction for the scientific community for research because of their ability to adapt to extreme habitats. Compatible solutes are among those factors which enable these microorganisms to thrive in such extreme habitats. Under osmotic stress, the majority of extremophiles accumulate specific organic solutes such as amino acids, sugars, polyols, and their derivatives. In addition, proteins in extremophiles are found to be evolved by changing their amino acid composition to alter the hydrophobicity of its core and surface charge to maintain activity. This chapter encompasses a comprehensive study about the role of various compatible solutes in the endurance of microorganisms under extremophilic conditions, synthesis of compatible solutes, nature of extremophilic proteins, and their applications. Furthermore, an attempt has been made to cover various strategies adopted by the scientific community while pursuing research on compatible solutes.

A Comprehensive Review on the Heavy Metal Toxicity and Sequestration in Plants

Heavy metal (HM) toxicity has become a global concern in recent years and is imposing a severe threat to the environment and human health. In the case of plants, a higher concentration of HMs, above a threshold, adversely affects cellular metabolism because of the generation of reactive oxygen species (ROS) which target the key biological molecules. Moreover, some of the HMs such as mercury and arsenic, among others, can directly alter the protein/enzyme activities by targeting their –SH group to further impede the cellular metabolism. Particularly, inhibition of photosynthesis has been reported under HM toxicity because HMs trigger the degradation of chlorophyll molecules by enhancing the chlorophyllase activity and by replacing the central Mg ion in the porphyrin ring which affects overall plant growth and yield. Consequently, plants utilize various strategies to mitigate the negative impact of HM toxicity by limiting the uptake of these HMs and their sequestration into the vacuoles with the help of various molecules including proteins such as phytochelatins, metallothionein, compatible solutes, and secondary metabolites. In this comprehensive review, we provided insights towards a wider aspect of HM toxicity, ranging from their negative impact on plant growth to the mechanisms employed by the plants to alleviate the HM toxicity and presented the molecular mechanism of HMs toxicity and sequestration in plants.

Role of Polyamines in Regulating Abiotic Stress Tolerance in Agricultural Crops: A Review

At present plants had to face adverse climatic fluctuations which are often detrimental for their growth in order to cope up with this situation, they develop certain adaptive tactics like synthesis of new proteins, micro RNAs, compatible solutes/ osmolites and radical release to avoid its adverse effects. Of these study on compatible solutes such as polyamines (PAs) gained popularity among researchers. In plant, they are involved in a wide variety of regulatory and cellular processes under normal conditions. During these stresses they acts by activating biosynthesis of signaling molecules like NO, H2O2; affects abscisic acid synthesis; Ca2+ homeostasis; ion channel signaling or even apoptosis in severe conditions. Over expression of PAs is reported in various stresses due to the presence of stress-responsive elements in the promoters of PA biosynthetic genes. This review paper, summarizes the effect of polyamines in boosting plants growth during abiotic stress and its mechanisms of action.

The MocR/GabR Ectoine and Hydroxyectoine Catabolism Regulator EnuR: Inducer and DNA Binding

The compatible solutes ectoine and 5-hydroxyectoine are widely synthesized by bacteria as osmostress protectants. These nitrogen-rich tetrahydropyrimidines can also be exploited as nutrients by microorganisms. Many ectoine/5-hydroxyectoine catabolic gene clusters are associated with a regulatory gene (enuR: ectoine nutrient utilization regulator) encoding a repressor protein belonging to the MocR/GabR sub-family of GntR-type transcription factors. Focusing on EnuR from the marine bacterium Ruegeria pomeroyi, we show that the dimerization of EnuR is mediated by its aminotransferase domain. This domain can fold independently from its amino-terminal DNA reading head and can incorporate pyridoxal-5′-phosphate (PLP) as cofactor. The covalent attachment of PLP to residue Lys302 of EnuR was proven by mass-spectrometry. PLP interacts with system-specific, ectoine and 5-hydroxyectoine-derived inducers: alpha-acetyldiaminobutyric acid (alpha-ADABA), and hydroxy-alpha-acetyldiaminobutyric acid (hydroxy-alpha-ADABA), respectively. These inducers are generated in cells actively growing with ectoines as sole carbon and nitrogen sources, by the EutD hydrolase and targeted metabolic analysis allowed their detection. EnuR binds these effector molecules with affinities in the low micro-molar range. Studies addressing the evolutionary conservation of EnuR, modelling of the EnuR structure, and docking experiments with the inducers provide an initial view into the cofactor and effector binding cavity. In this cavity, the two high-affinity inducers for EnuR, alpha-ADABA and hydroxy-alpha-ADABA, are positioned such that their respective primary nitrogen group can chemically interact with PLP. Purified EnuR bound with micro-molar affinity to a 48 base pair DNA fragment containing the sigma-70 type substrate-inducible promoter for the ectoine/5-hydroxyectoine importer and catabolic gene cluster. Consistent with the function of EnuR as a repressor, the core elements of the promoter overlap with two predicted EnuR operators. Our data lend themselves to a straightforward regulatory model for the initial encounter of EnuR-possessing ectoine/5-hydroxyectoine consumers with environmental ectoines and for the situation when the external supply of these compounds has been exhausted by catabolism.

Examining the Osmotic Response of Acidihalobacter aeolianus after Exposure to Salt Stress

Acidihalobacter aeolianus is an acidophilic, halo-tolerant organism isolated from a marine environment near a hydrothermal vent, an ecosystem whereby levels of salinity and total dissolved salts are constantly fluctuating creating ongoing cellular stresses. In order to survive these continuing changes, the synthesis of compatible solutes—also known as organic osmolytes—is suspected to occur, aiding in minimising the overall impact of environmental instability. Previous studies on A. aeolianus identified genes necessary for the accumulation of proline, betaine and ectoine, which are known to act as compatible solutes in other halophilic species. In this study, the impact of increasing the osmotic stress as well as the toxic ion effect was investigated by subjecting A. aeolianus to concentrations of NaCl and MgSO4 up to 1.27 M. Exposure to high concentrations of Cl- resulted in the increase of ectC expression in log-phase cells with a corresponding accumulation of ectoine at stationary phase. Osmotic stress via MgSO4 exposure did not trigger the same up-regulation of ectC or accumulation of ectoine, indicating the transcriptionally regulated response against osmotic stress was induced by chloride toxicity. These findings have highlighted how the adaptive properties of halo-tolerant organisms in acidic environments are likely to differ and are dependent on the initial stressor.

Adaptation Strategies to Improve the Resistance of Oilseed Crops to Heat Stress Under a Changing Climate: An Overview

Temperature is one of the decisive environmental factors that is projected to increase by 1. 5°C over the next two decades due to climate change that may affect various agronomic characteristics, such as biomass production, phenology and physiology, and yield-contributing traits in oilseed crops. Oilseed crops such as soybean, sunflower, canola, peanut, cottonseed, coconut, palm oil, sesame, safflower, olive etc., are widely grown. Specific importance is the vulnerability of oil synthesis in these crops against the rise in climatic temperature, threatening the stability of yield and quality. The natural defense system in these crops cannot withstand the harmful impacts of heat stress, thus causing a considerable loss in seed and oil yield. Therefore, a proper understanding of underlying mechanisms of genotype-environment interactions that could affect oil synthesis pathways is a prime requirement in developing stable cultivars. Heat stress tolerance is a complex quantitative trait controlled by many genes and is challenging to study and characterize. However, heat tolerance studies to date have pointed to several sophisticated mechanisms to deal with the stress of high temperatures, including hormonal signaling pathways for sensing heat stimuli and acquiring tolerance to heat stress, maintaining membrane integrity, production of heat shock proteins (HSPs), removal of reactive oxygen species (ROS), assembly of antioxidants, accumulation of compatible solutes, modified gene expression to enable changes, intelligent agricultural technologies, and several other agronomic techniques for thriving and surviving. Manipulation of multiple genes responsible for thermo-tolerance and exploring their high expressions greatly impacts their potential application using CRISPR/Cas genome editing and OMICS technology. This review highlights the latest outcomes on the response and tolerance to heat stress at the cellular, organelle, and whole plant levels describing numerous approaches applied to enhance thermos-tolerance in oilseed crops. We are attempting to critically analyze the scattered existing approaches to temperature tolerance used in oilseeds as a whole, work toward extending studies into the field, and provide researchers and related parties with useful information to streamline their breeding programs so that they can seek new avenues and develop guidelines that will greatly enhance ongoing efforts to establish heat stress tolerance in oilseeds.

Triacontanol modulates salt stress tolerance in cucumber by altering the physiological and biochemical status of plant cells

Cucumber is an important vegetable but highly sensitive to salt stress. The present study was designed to investigate the comparative performance of cucumber genotypes under salt stress (50 mmol L−1) and stress alleviation through an optimized level of triacontanol @ 0.8 mg L−1. Four cucumber genotypes were subjected to foliar application of triacontanol under stress. Different physiological, biochemical, water relations and ionic traits were observed to determine the role of triacontanol in salt stress alleviation. Triacontanol ameliorated the lethal impact of salt stress in all genotypes, but Green long and Marketmore were more responsive than Summer green and 20252 in almost all the attributes that define the genetic potential of genotypes. Triacontanol performs as a good scavenger of ROS by accelerating the activity of antioxidant enzymes (SOD, POD, CAT) and compatible solutes (proline, glycinebetaine, phenolic contents), which lead to improved gas exchange attributes and water relations and in that way enhance the calcium and potassium contents or decline the sodium and chloride contents in cucumber leaves. Furthermore, triacontanol feeding also shows the answer to yield traits of cucumber. It was concluded from the results that the salinity tolerance efficacy of triacontanol is valid in enhancing the productivity of cucumber plants under salt stress. Triacontanol was more pronounced in green long and marketer green than in summer green and 20252. Hence, the findings of this study pave the way towards the usage of triacontanol @ 0.8 mg L−1, and green long and marketer genotypes may be recommended for saline soil.

Optimization of compatible solutes for improving survival of freeze-dried Lactobacillus delbrueckii subsp. bulgaricus using Box-Behnken design

Lactobacillus delbrueckii subsp. bulgaricus is widely used in yogurt as a starter. The freeze-drying process may cause bacteria death. In the present work, the effect of three solutes (NaCl, sorbitol, and sodium glutamate) in MRS on viability of L.bulgaricus during freeze-drying was investigated. The optimal combination of adequate solutes was chosen by Box-Behnken Design. The survival rate and viable counts in freeze-dried powder, as well as the viable counts in broth, were used as responses. The results revealed that the optimum combination of solutes in MRS broth were 0.50% NaCl, 0.19% sorbitol, and 0.06% sodium glutamate. Under these optimal conditions, the survival rate was 53.2±0.14%, the viable counts in freeze-dried powder was 8.51±0.23×1010 CFU/g, and the viable counts in broth was 6.05±0.19 ×108 CFU/mL, which were increased by 17.18%, 15.94%, and 17.31%, respectively, compared to the control. This research demonstrated the possibility of viability improvement of L.bulgaricus, which may provide a feasible reference for industrial development.

Molecular control to salt tolerance mechanisms of woody plants: recent achievements and perspectives

Key message Woody plants have salt-tolerant mechanisms similar to those developed by non-woody plants. Among others, compartmentalization of ions, production of compatible solutes, synthesis of specific proteins and metabolites, and induction of transcriptional factors are the most relevant. Woody plant-associated microbial interactions as well as naturally stress-adapted trees are resources that deserve to be deepened to fully understand the tolerance mechanisms. Context The high variability of salinity responses found in woody plants implies a high potentiality for germplasm selection and breeding. Salt tolerance mechanisms of plants are regulated by numerous genes, which control ion homeostasis, production of compatible solutes and specific proteins, and activation or repression of specific transcription factors. Despite the fact that numerous studies have been done on herbaceous model plants, knowledge about salt tolerance mechanisms in woody plants is still scarce. Aims The present review critically evaluates molecular control of salt tolerance mechanisms of woody plants, focusing on the regulation and compartmentalization of ions, production of compatible solutes, activation of transcription factors, and differential expression of stress response-related proteins, including omics-based approaches and the role of plant-microbial interactions. The potential identification of genes from naturally stress-adapted woody plants and the integration of the massive omics data are also discussed. Conclusion In woody plants, salt tolerance mechanisms seem not to diverge to those identified in non-woody plants. More comparative studies between woody and non-woody salt tolerance plants will be relevant to identify potential molecular mechanisms specifically developed for wood plants. In this sense, the activation of metabolic pathways and molecular networks by novel genetic engineering techniques is key to establish strategies to improve the salt tolerance in woody plant species and to contribute to more sustainable agricultural and forestry systems.