Tomato UDP-glucose sterol glycosyltransferases: A family of developmental and stress regulated genes that encode cytosolic and membrane-associated forms of the enzyme

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

Withanolides: Elucidating steroidal lactone biosynthesis in Nightshades

Withania somnifera (Solanaceae) is well known in ayurvedic medicine as a strengthening tonic for various medical purposes. Its effects are mainly due to withanolides, a class of steroidal lactones with diverse oxidation patterns present in various nightshade plants. Pharmacological studies attributed anti-proliferative and anti-inflammatory properties to withanolides. However, obtaining medicinally active withanolides can be complicated, as complex mixtures are present in producing plants and total synthesis of withanolides is costly and time consuming. Therefore, investigation of their biosynthesis is important to enable biotechnological enhancement and to provide novel insights into plant steroid biochemistry. This work aimed to investigate withanolide biosynthesis in Physalis peruviana and Withania somnifera. Both plants were investigated for their main withanolides, as producers can accumulate a diverse array of withanolides, depending on the cultivation conditions. Here, besides several known withanolides, two yet unknown, truncated withanolides (irinan A (1) and B (2)) were isolated from P. peruviana and their structures were elucidated. As intermediates of withanolide biosynthesis were needed for enzyme assays but are neither known, nor commercially available, metabolic engineering in yeast was attempted to divert yeast ergosterol biosynthesis towards production of 24-methyldesmosterol (3), the last known precursor in withanolide biosynthesis. However, while production of the precursor 24-methylenecholesterol (4) was temporarily observed, 3 did not accumulate. Furthermore, based on the biosynthetic hypothesis, 21 cytochrome P450 (P450) and 14 dehydratase (DH) gene candidates were selected after analysis of three withanolide-producing species. Of those, 17 P450 and 6 DH candidates could be cloned and evaluated by gene silencing in W. somnifera, identifying 5 P450 and 2 DH gene candidates where silencing evoked significant decrease of the main withanolide (withaferin A, 5). Those candidates were further examined by heterologous expression experiments in the model plant Nicotiana benthamiana. Here activity on the substrate 24-methyldesmosterol was detected for one candidate (P450-7), while another exhibited activity on native cycloartenol (6) from the host plant (P450-17). Further investigation of P450-17 revealed that orthologs were present in tomato and potato, both non-producers of withanolides. In both plants P450-17 homologous genes are arranged in gene clusters, with neither the genes nor the cluster being reported before. In conclusion, this work provides insights into oxidations involved in withanolide biosynthesis and yet unknown phytosterol pathways in Solanaceae plants

Tomato STEROL GLYCOSYLTRANSFERASE 1 silencing unveils a major role of steryl glycosides in plant and fruit development

Free and glycosylated sterols localize in the plant cell plasma membrane, where in combination with other lipids regulate its structure and function. The role of glycosylated sterols in regulating membrane-associated biological processes is more relevant in plants like tomato (Solanum lycopersicum), in which glycosylated sterols are the predominant sterols. A proper ratio of free sterols versus glycosylated sterols has proven to be essential for proper plant performance in several species, but almost nothing is known in tomato. To assess the role of glycosylated sterols in tomato plant and fruit development, we generated transgenic lines of tomato cultivar Micro-Tom expressing two different amiRNAs devised to silence STEROL GLYCOSYLTRANSFERASE 1, the most actively expressed of the four genes encoding sterol glycosyltransferases in this plant. STEROL GLYCOSYLTRANSFERASE 1 gene silencing caused moderate plant dwarfism and reduced fruit size. Analysis of the profile of glycosylated sterols throughout fruit development demonstrated that the maintenance of proper levels of these compounds during the early stages of fruit development is essential for normal fruit growth, since reduced levels of glycosylated sterols trigger a transcriptional downregulatory response that affects genes involved in processes that are critical for proper fruit development, such as seed filling, cell wall extension and auxin signaling

Transcriptome and metabolite analyses in Azadirachta indica: identification of genes involved in biosynthesis of bioactive triterpenoids

Bhambhani S, Lakhwani D, Gupta P, et al. Transcriptome and metabolite analyses in Azadirachta indica: identification of genes involved in biosynthesis of bioactive triterpenoids. SCIENTIFIC REPORTS. 2017;7(1): 5043.Azadirachta indica A. Juss, commonly known as Neem, is the reservoir of triterpenoids of economic importance. Metabolite analysis of different developmental stages of leaf and fruit suggests tissue-specific accumulation of the major triterpenoids in this important tree. Though biosynthesis of these complex molecules requires substrate flux from the isoprenoid pathway, enzymes involved in late biosynthetic steps remain uncharacterized. We established and analyzed transcriptome datasets from leaf and fruit and identified members of gene families involved in intermediate steps of terpenoid backbone biosynthesis and those related to secondary transformation leading to the tissue-specific triterpenoid biosynthesis. Expression analysis suggests differential expression of number of genes between leaf and fruit and probable participation in the biosynthesis of fruit-specific triterpenoids. Genome-wide analysis also identified members of gene families putatively involved in secondary modifications in late biosynthetic steps leading to the synthesis of highly oxygenated triterpenoids. Expression and molecular docking analyses suggest involvement of specific members of CYP450 family in secondary modifications for the biosynthesis of bioactive triterpenoids. This study generated rich genomic resource and identified genes involved in biosynthesis of important molecules, which will aid in the advancement of tools for functional genomics and elucidation of the biosynthesis of triterpenoid from this important tree

Tomato STEROL GLYCOSYLTRANSFERASE 1 silencing unveils a major role of steryl glycosides in plant and fruit development

Free and glycosylated sterols localize in the plant cell plasma membrane, where in combination with other lipids regulate its structure and function. The role of glycosylated sterols in regulating membrane-associated biological processes is more relevant in plants like tomato (Solanum lycopersicum), in which glycosylated sterols are the predominant sterols. A proper ratio of free sterols versus glycosylated sterols has proven to be essential for proper plant performance in several species, but almost nothing is known in tomato. To assess the role of glycosylated sterols in tomato plant and fruit development, we generated transgenic lines of tomato cultivar Micro-Tom expressing two different amiRNAs devised to silence STEROL GLYCOSYLTRANSFERASE 1, the most actively expressed of the four genes encoding sterol glycosyltransferases in this plant. STEROL GLYCOSYLTRANSFERASE 1 gene silencing caused moderate plant dwarfism and reduced fruit size. Analysis of the profile of glycosylated sterols throughout fruit development demonstrated that the maintenance of proper levels of these compounds during the early stages of fruit development is essential for normal fruit growth, since reduced levels of glycosylated sterols trigger a transcriptional downregulatory response that affects genes involved in processes that are critical for proper fruit development, such as seed filling, cell wall extension and auxin signaling