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.

Exogenous Glycine Betaine Application Improves Freezing Tolerance of Cabbage (Brassica oleracea L.) Leaves

Exogenous glycine betaine (GB) application has been reported to improve plant tolerance to various abiotic stresses, but its effect on freezing tolerance has not been well studied. We investigated the effect of exogenous GB on freezing tolerance of cabbage (Brassica oleracea L.) leaves. Seedlings fed with 30 mM GB via sub-irrigation showed effectively assimilated GB as evident by higher GB concentration. Exogenous GB did not retard leaf-growth (fresh weight, dry weight, and leaf area) rather slightly promoted it. Temperature controlled freeze-thaw tests proved GB-fed plants were more freeze-tolerant as indicated by lower electrolyte leakage (i.e., indication of less membrane damage) and alleviating oxidative stress (less accumulation of O2•− and H2O2, as well as of malondialdehyde (MDA)) following a relatively moderate or severe freeze-thaw stress, i.e., −2.5 and −3.5 °C. Improved freezing tolerance induced by exogenous GB application may be associated with accumulation of compatible solute (proline) and antioxidant (glutathione). GB-fed leaves also had higher activity of antioxidant enzymes, catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD). These changes, together, may improve freezing tolerance through membrane protection from freeze-desiccation and alleviation of freeze-induced oxidative stress.

Drought mitigation in cocoa (Theobroma cacao L.) through developing tolerant hybrids

Background Cocoa, being a shade loving crop cannot withstand long periods of water stress. Breeding for drought tolerance is the need of the hour due to change in climatic condition and extension of crop to non-traditional areas. Hybrids were produced by crossing four tolerant genotypes in all possible combination. The cross GV1 55 x M 13.12 didn’t yield any fruit due to cross incompatibility between these genotypes. Various biochemical parameters act as the true indicators to select tolerant and susceptible types. The major biochemical parameters considered after imposing stress included proline, nitrate reductase activity, superoxide dismutase content and glycine betaine. Results The drought tolerant hybrids were having high amount of proline, superoxide dismutase enzyme and glycine betaine content. Normally, plants having drought stress show low amount of nitrate reductase activity. However, in case of hybrids, the drought tolerant hybrids were having higher NR activity than the susceptible hybrids. The highest amount of NR was found in the control plants kept at fully irrigated conditions. Conclusions This experiment showed the role of different biochemical enzymes and osmolytes in giving tolerance to plants during drought stress. Logistic regression analysis selected proline and nitrate reductase as the two biochemical markers for identifying efficient drought tolerant genotypes in the future breeding programmes.

Insights into Metabolic Engineering of the Biosynthesis of Glycine Betaine and Melatonin to Improve Plant Abiotic Stress Tolerance

Metabolic engineering in plant can be describe as a tool using molecular biological technologies which promotes enzymatic reactions that can enhance the biosynthesis of existing compounds such as glycine betaine (GB) in plant species that are able to accumulate GB, or produce news compounds like GB in non-accumulators plants. Moreover we can include to these definition, the mediation in the degradation of diverse compounds in plant organism. For decades, one of the most popular ideas in metabolic engineering literature is the idea that the improvement of gly betaine or melatonin accumulation in plant under environmental stress can be the main window to ameliorate stress tolerance in diverse plant species. A challenging problem in this domain is the integration of different molecular technologies like transgenesis, enzyme kinetics, promoter analysis, biochemistry and genetics, protein sorting, cloning or comparative physiology to reach that objective. A large number of approaches have been developed over the last few decades in metabolic engineering to overcome this problem. Therefore, we examine some previous work and propose some understanding about the use of metabolic engineering in plant stress tolerance. Moreover, this chapter will focus on melatonin (Hormone) and gly betaine (Osmolyte) biosynthesis pathways in engineering stress resistance.