Improving water deficit tolerance of Salvia officinalis L. using putrescine

To study the effects of foliar application of putrescine (distilled water (0), 0.75, 1.5, and 2.25 mM) and water deficit stress (20%, 40%, 60%, and 80% available soil water depletion (ASWD)) on the physiological, biochemical, and molecular attributes of sage, a factorial experiment was performed in a completely randomized design with three replications in the growth chamber. The results of qRT-PCR analysis showed that putrescine concentration, irrigation regime, and the two-way interaction between irrigation regime and putrescine concentration significantly influenced cineole synthase, sabinene synthase, and bornyl diphosphate synthase relative expression. The highest concentration of 1,8-cineole, camphor, α-thujone, β-thujone, cineole synthase, sabinene synthase, and bornyl diphosphate synthase were obtained in the irrigation regime of 80% ASWD with the application of 0.75 mM putrescine. There was high correlation between expression levels of the main monoterpenes synthase and the concentration of main monoterpenes. The observed correlation between the two enzyme activities of APX and CAT strongly suggests they have coordinated action. On the other hand, the highest PO and SOD concentrations were obtained with the application of 0.75 mM putrescine under the irrigation regime of 40% ASWD. Putrescine showed a significant increase in LAI and RWC under water deficit stress. There was an increasing trend in endogenous putrescine when putrescine concentration was increased in all irrigation regimes. Overall, the results suggest that putrescine may act directly as a stress-protecting compound and reduced H2O2 to moderate the capacity of the antioxidative system, maintain the membrane stability, and increase secondary metabolites under water deficit stress.

Collaborative subcellular compartmentalization to improve GPP utilization and boost sabinene accumulation in Saccharomyces cerevisiae

Background Monoterpenes and their derivatives play an important role as flavorings, perfume additives, pharmaceuticals and advanced biofuels. GPP (geranyl diphosphate) is the direct precursor to biosynthesize this class of products. The present study for monoterpenes synthesis in yeast focused on manipulation of metabolic flux to improve GPP supply. However, if the subcellular distribution of GPP and monoterpene synthase were not coincided with each other, it would be hard to well utilize GPP, leading to a waste of carbon sources.Results Herein, we took sabinene production in Saccharomyces cerevisiae as an instance, and confirmed the location of N-truncated sabinene synthase (t34SabS1) in yeast cytosol (C). We also revealed the existence of GPP pools in organelles [such as lipid monolayer membrane-bound peroxisomes (P) and bilayer membrane-bound mitochondria (M)] beside cytosol. In order to minimize the loss of GPP, an engineering strategy was proposed to coordinated compartmentalization of sabinene synthase. Initially, expression of t34SabS1 in an ERG20-downregulated host only obtained 19.4 mg/L sabinene. Combined targeting t34SabS1 into CM (cytosol and mitochondria) increased sabinene production to 64.6 mg/L. This titer was significantly higher than those generated by harnessing other combinations of subcellular locations ( i.e. CP, PM and even CPM). Further overexpression of the genes involved in mitochondria morphology uncovered four novel molecular targets ( i.e. FIS1 , LSB3 , MBA1 and AIM25 ) associating with sabinene output. Especially, overexpression of AIM25 enhanced the sabinene production to 90.4 mg/L. Eventually, integrating all above engineered genes into host chromosome achieved 154.9 mg/L of sabinene.Conclusions The engineering approaches of this study improve GPP utilization and boost sabinene accumulation almost 60-fold of the original titer. This research highlights the strategy of organelle engineering to improve precursor utilization and to enhance the compartmentalized pathway. It also sets a good reference to synthesize other valuable monoterpenes and their derivatives in eukaryotic hosts.

Transcriptomic and metabolomic analyses provide insight into the volatile compounds of citrus leaves and flowers

Background Previous reports have mainly focused on the volatiles in citrus fruits, and there have been few reports about the volatiles in citrus leaves and flowers. However, citrus leaves and flowers are also rich in volatile compounds with unique aromas. Here, to investigate the volatiles in citrus leaves and flowers, volatile profiling was performed on leaves from 62 germplasms and flowers from 25 germplasms. Results In total, 196 and 82 volatile compounds were identified from leaves of 62 citrus germplasms and flowers of 25 citrus germplasms, respectively. The dominant volatile terpenoids were more diverse in citrus leaves than in peels. A total of 34 volatile terpenoids were commonly detected in the leaves of at least 20 germplasms, among which 31 were overaccumulated in the leaves of wild or semiwild germplasms. This result was consistent with the high expression levels of five genes and one key gene of the mevalonate and 2-C-methyl-D-erythritol-4-phosphate (MEP) biosynthetic pathways, respectively, as well as the low expression levels of geranylgeranyl diphosphate synthase of the MEP pathway, relative to the levels in cultivars. Fully open flowers showed increased levels of four terpene alcohols and a decrease in sabinene content compared with balloon-stage flowers, especially in sweet orange. A monoterpene synthase gene was identified and functionally characterized as a sabinene synthase in vitro. Conclusions Collectively, our results suggest that 31 important terpenoids are abundant in wild or semiwild citrus germplasms, possibly because of a negative effect of domestication on the volatiles in citrus leaves. The sweet smell of fully open flowers may be attributed to increased levels of four terpene alcohols. In addition, a sabinene synthase gene was identified by combined transcriptomic and metabolomic analyses.