A family of methyl esterases converts methyl salicylate to salicylic acid in ripening tomato fruit.

Methyl salicylate imparts a potent flavor and aroma described as medicinal and wintergreen that is undesirable in tomato (Solanum lycopersicum) fruit. Plants control the quantities of methyl salicylate through a variety of biosynthetic pathways, including the methylation of salicylic acid to form methyl salicylate and subsequent glycosylation to prevent methyl salicylate emission. Here, we identified a subclade of tomato methyl esterases, SALICYLIC ACID METHYL ESTERASE1-4, responsible for demethylation of methyl salicylate to form salicylic acid in fruits. This family was identified by proximity to a highly significant methyl salicylate genome-wide association study locus on chromosome 2. Genetic mapping studies in a biparental population confirmed a major methyl salicylate locus on chromosome 2. Fruits from SlMES1 knockout lines emitted significantly (P < 0,05, t test) higher amounts of methyl salicylate than wild-type fruits. Double and triple mutants of SlMES2, SlMES3, and SlMES4 emitted even more methyl salicylate than SlMES1 single knockouts-but not at statistically distinguishable levels-compared to the single mutant. Heterologously expressed SlMES1 and SlMES3 acted on methyl salicylate in vitro, with SlMES1 having a higher affinity for methyl salicylate than SlMES3. The SlMES locus has undergone major rearrangement, as demonstrated by genome structure analysis in the parents of the biparental population. Analysis of accessions that produce high or low levels of methyl salicylate showed that SlMES1 and SlMES3 genes expressed the highest in the low methyl salicylate lines. None of the MES genes were appreciably expressed in the high methyl salicylate-producing lines. We concluded that the SlMES gene family encodes tomato methyl esterases that convert methyl salicylate to salicylic acid in ripe tomato fruit. Their ability to decrease methyl salicylate levels by conversion to salicylic acid is an attractive breeding target to lower the level of a negative contributor to flavor.

A single promoter-TALE system for tissue-specific and tuneable expression of multiple genes in rice.

In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems-dTALE1-STAP1 and dTALE2-STAP2-can activate STAP-driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell-types, requiring empirical evaluation in each case. dTALE expression leads to off-target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE-STAP system thus provides a powerful approach to fine-tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.

Comparative transcriptome analyses shed light on carotenoid production and plastid development in melon fruit.

Carotenoids, such as ß-carotene, accumulate in chromoplasts of various fleshy fruits, awarding them with colors, aromas, and nutrients. The Orange (CmOr) gene controls ß-carotene accumulation in melon fruit by posttranslationally enhancing carotenogenesis and repressing ß-carotene turnover in chromoplasts. Carotenoid isomerase (CRTISO) isomerizes yellow prolycopene into red lycopene, a prerequisite for further metabolism into ß-carotene. We comparatively analyzed the developing fruit transcriptomes of orange-colored melon and its two isogenic EMS-induced mutants, low-ß (Cmor) and yofi (Cmcrtiso). The Cmor mutation in low-ß caused a major transcriptomic change in the mature fruit. In contrast, the Cmcrtiso mutation in yofi significantly changed the transcriptome only in early fruit developmental stages. These findings indicate that melon fruit transcriptome is primarily altered by changes in carotenoid metabolic flux and plastid conversion, but minimally by carotenoid composition in the ripe fruit. Clustering of the differentially expressed genes into functional groups revealed an association between fruit carotenoid metabolic flux with the maintenance of the photosynthetic apparatus in fruit chloroplasts. Moreover, large numbers of thylakoid localized photosynthetic genes were differentially expressed in low-ß. CmOR family proteins were found to physically interact with light-harvesting chlorophyll a-b binding proteins, suggesting a new role of CmOR for chloroplast maintenance in melon fruit. This study brings more insights into the cellular and metabolic processes associated with fruit carotenoid accumulation in melon fruit and reveals a new maintenance mechanism of the photosynthetic apparatus for plastid development.

Exploring eukaryotic formate metabolisms to enhance microbial growth and lipid accumulation.

BACKGROUND: C1 substrates (such as formate and methanol) are promising feedstock for biochemical/biofuel production. Numerous studies have been focusing on engineering heterologous pathways to incorporate C1 substrates into biomass, while the engineered microbial hosts often demonstrate inferior fermentation performance due to substrate toxicity, metabolic burdens from engineered pathways, and poor enzyme activities. Alternatively, exploring native C1 pathways in non-model microbes could be a better solution to address these challenges. RESULTS: An oleaginous fungus, Umbelopsis isabellina, demonstrates an excellent capability of metabolizing formate to promote growth and lipid accumulation. By co-feeding formate with glucose at a mole ratio of 3.9:1, biomass and lipid productivities of the culture in 7.5 L bioreactors were improved by 20 and 70%, respectively. 13C-metabolite analysis, genome annotations, and enzyme assay further discovered that formate not only provides an auxiliary energy source [promoting NAD(P)H and ATP] for cell anabolism, but also contributes carbon backbones via folate-mediated C1 pathways. More interestingly, formate addition can tune fatty acid profile and increase the portion of medium-chain fatty acids, which would benefit conversion of fungal lipids for high-quality biofuel production. Flux balance analysis further indicates that formate co-utilization can power microbial metabolism to improve biosynthesis, particularly on glucose-limited cultures. CONCLUSION: This study demonstrates Umbelopsis isabellina's strong capability for co-utilizing formate to produce biomass and enhance fatty acid production. It is a promising non-model platform that can be potentially integrated with photochemical/electrochemical processes to efficiently convert carbon dioxide into biofuels and value-added chemicals.