Tomato UDP-Glucose Sterol Glycosyltransferases: A Family of Developmental and Stress Regulated Genes that Encode Cytosolic and Membrane-Associated Forms of the Enzyme

Abstract

Sterol glycosyltransferases (SGTs) catalyze the glycosylation of the free hydroxyl group at C-3 position of sterols to produce sterol glycosides. Glycosylated sterols and free sterols are primarily located in cell membranes where in combination with other membrane-bound lipids play a key role in modulating their properties and functioning. In contrast to most plant species, those of the genus Solanum contain very high levels of glycosylated sterols, which in the case of tomato may account for more than 85% of the total sterol content. In this study, we report the identification and functional characterization of the four members of the tomato (Solanum lycopersicum cv. Micro-Tom) SGT gene family. Expression of recombinant SlSGT proteins in E. coli cells and N. benthamiana leaves demonstrated the ability of the four enzymes to glycosylate different sterol species including cholesterol, brassicasterol, campesterol, stigmasterol, and β-sitosterol, which is consistent with the occurrence in their primary structure of the putative steroid-binding domain found in steroid UDP-glucuronosyltransferases and the UDP-sugar binding domain characteristic for a superfamily of nucleoside diphosphosugar glycosyltransferases. Subcellular localization studies based on fluorescence recovery after photobleaching and cell fractionation analyses revealed that the four tomato SGTs, like the Arabidopsis SGTs UGT80A2 and UGT80B1, localize into the cytosol and the PM, although there are clear differences in their relative distribution between these two cell fractions. The SlSGT genes have specialized but still largely overlapping expression patterns in different organs of tomato plants and throughout the different stages of fruit development and ripening. Moreover, they are differentially regulated in response to biotic and abiotic stress conditions. SlSGT4 expression increases markedly in response to osmotic, salt, and cold stress, as well as upon treatment with abscisic acid and methyl jasmonate. Stress-induced SlSGT2 expression largely parallels that of SlSGT4. On the contrary, SlSGT1 and SlSGT3 expression remains almost unaltered under the tested stress conditions. Overall, this study contributes to broaden the current knowledge on plant SGTs and provides support to the view that tomato SGTs play overlapping but not completely redundant biological functions involved in mediating developmental and stress responses.

Emerging Applications of Fermented Fruit Enzyme Beverages as Nutraceutical, Functional Food, and Designer Food Products: A Review

“…Sterol glycosyltransferases (SGTs) catalyze the glycosylation of the free hydroxyl group at C-3 position of sterols to produce sterol glycosides. Expression of recombinant SlSGT proteins in E. coli cells and N. benthamiana leaves demonstrated the ability of the four enzymes to glycosylate different sterol species including cholesterol, brassicasterol, campesterol, stigmasterol, and β-sitosterol, which is consistent with the occurrence in their primary structure of the putative steroid-binding domain found in steroid UDP-glucuronosyltransferases and the UDP-sugar binding domain characteristic for a superfamily of nucleoside diphosphosugar glycosyltransferases 60 . The importance of the P450 enzyme CYP1B1 in both cancer therapy and prevention are reviewed and evidence is discussed, which provides strong biological plausibility for the therapeutic merits of this approach.…”

Identification and Characterization of Sterol Acyltransferases Responsible for Steryl Ester Biosynthesis in Tomato

“…Micro-Tom plants were grown in pots filled with a mixture of peat (Klasmann TS2), vermiculite, and perlite (2:1:1, v/v/v) at 25°C in a greenhouse. To grow seedlings under axenic conditions, seeds were sterilized as described by Ramírez-Estrada et al (2017) and sown in glass jars containing 0.5× Murashige and Skoog (MS) basal salts medium (pH 5.8) supplemented with Gamborg B5 vitamins and sucrose 3% (w/v), and solidified with 0.8% (w/v) plant agar. Jars were kept in darkness at 24°C for 2 days and transferred to a growth chamber under a 16 h light/8 h dark illumination regime (150 μE m -2 s -1 intensity) at 24°C.…”

“…The coding regions of AtASAT1, SlASAT1, and SlPSAT1 were amplified by PCR using high-fidelity AccuPrime Taq DNA polymerase (Invitrogen), specific primer pairs encompassing the corresponding start and stop codons (Supplementary Table 1 ), and cDNA synthesized from total RNA obtained from A. thaliana leaves or tomato red fruit pericarp. RNA isolation and cDNA synthesis were performed as described previously ( Ramírez-Estrada et al, 2017 ). The CACC sequence was added to the 5′ end of the forward primers to facilitate directional insertion of the amplified sequences into the pENTR/D-TOPO vector by Gateway recombination-based cloning (TOPO cloning kit, Invitrogen).…”

“…The cDNA samples for RT-qPCR gene expression analysis were prepared from DNA-free total RNA samples obtained as indicated above. Real-time PCR assays were performed in triplicate with SYBR Green I Master (Roche Diagnostics) using a Light Cycler 480 detection system (Roche Diagnostics), essentially as described by Ramírez-Estrada et al (2017) . Specific primer pairs were designed for SlPSAT1 and SlASAT1 (Supplementary Table 1 ).…”

Regional differences in the abiotic environment contribute to genomic divergence within a wild tomato species

“…SGT genes have been shown to have functional roles in abiotic stress tolerance in several species (Griebel & Zeier, 2010;Hugly et al, 1990;Kumar et al, 2015;Senthil-Kumar et al, 2013). Expression of SGT4 and its paralog SGT2 is rapidly induced upon exposure to cold and salt stress in domesticated tomato (Ramirez-Estrada et al, 2017). In Arabidopsis thaliana SGT knock-TA B L E 3 Annotated gene models located within 10kb of the top 15 outlier SNPs along the first RDA axis, after accounting for spatial structure.…”