Ectopic overexpression of WsSGTL1, a sterol glucosyltransferase gene in Withania somnifera, promotes growth, enhances glycowithanolide and provides tolerance to abiotic and biotic stresses

Abstract

Overexpression of sterol glycosyltransferase (SGTL1) gene of Withania somnifera showing its involvement in glycosylation of withanolide that leads to enhanced growth and tolerance to biotic and abiotic stresses. Withania somnifera is widely used in Ayurvedic medicines for over 3000 years due to its therapeutic properties. It contains a variety of glycosylated steroids called withanosides that possess neuroregenerative, adaptogenic, anticonvulsant, immunomodulatory and antioxidant activities. The WsSGTL1 gene specific for 3β-hydroxy position has a catalytic specificity to glycosylate withanolide and sterols. Glycosylation not only stabilizes the products but also alters their physiological activities and governs intracellular distribution. To understand the functional significance and potential of WsSGTL1 gene, transgenics of W. somnifera were generated using Agrobacterium tumefaciens-mediated transformation. Stable integration and overexpression of WsSGTL1 gene were confirmed by Southern blot analysis followed by quantitative real-time PCR. The WsGTL1 transgenic plants displayed number of alterations at phenotypic and metabolic level in comparison to wild-type plants which include: (1) early and enhanced growth with leaf expansion and increase in number of stomata; (2) increased production of glycowithanolide (majorly withanoside V) and campesterol, stigmasterol and sitosterol in glycosylated forms with reduced accumulation of withanolides (withaferin A, withanolide A and withanone); (3) tolerance towards biotic stress (100 % mortality of Spodoptera litura), improved survival capacity under abiotic stress (cold stress) and; (4) enhanced recovery capacity after cold stress, as indicated by better photosynthesis performance, chlorophyll, anthocyanin content and better quenching regulation of PSI and PSII. Our data demonstrate overexpression of WsSGTL1 gene which is responsible for increase in glycosylated withanolide and sterols, and confers better growth and tolerance to both biotic and abiotic stresses.

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

“…UGT80A2 activity seems to account for most of the sitosteryl and stigmasteryl glucoside production in seeds while UGT80B1 preferentially forms brassicasteryl glucosides (DeBolt et al, 2009; Stucky et al, 2015). It is also worth noting that some plant SGTs seem to have broad substrate specificity, as exemplified by the SGTs of W. somnifera , a Solanaceae species that contains a variety of steroidal aglycones (withanolides), which can be glycosylated along with sterols by members of the WsSGT family (Saema et al, 2016; Singh et al, 2016). WsSGTs have been predicted to interact with a variety of sterols and withanolides but with different substrate preferences.…”

“…The stress-induced expression of specific tomato SlSGT genes is also consistent with the response to abiotic stress of mutant plants with altered levels of SGTs. Thus, an Arabidopsis T-DNA insertion mutant defective in the UGT80B1 gene shows increased sensitivity to cold and heat stress as compared to wild-type plants (Mishra et al, 2015), whereas constitutive expression of WsSGTL1 in Arabidopsis results in enhanced cold, heat, and salt tolerance (Mishra et al, 2013) and overexpression of the same enzyme in W. somnifera and tobacco leads to enhanced tolerance to cold and salt, respectively (Pandey et al, 2014; Saema et al, 2016). Interestingly, the expression of SlSGT2 and SlSGT4 , but not that of SlSGT1 and SlSGT3 , also increased markedly after 3 h of treatment with ABA ( Figure 9A ), a phytohormone involved in the response to most abiotic stresses (Finkelstein, 2013).…”

“…These results, which are different to those observed in W. somnifera where the expression of all the SGT genes was enhanced by SA (Chaturvedi et al, 2012), suggest that in tomato SGs might be involved in JA-mediated defense rather than in SA-dependent pathways, while both would be involved in W. somnifera (Chaturvedi et al, 2012). It remains to be established whether the differential expression pattern of SGT genes in response to MeJA and SA in tomato and W. somnifera might be related to differences in substrate specificity between the SGTs of these two species, since W. somnifera SGTs also glycosylate withanolides in addition to sterols (Saema et al, 2016; Singh et al, 2016). Altogether, these results suggest that, at least in tomato, SGs are involved in plant response to abiotic stress mediated by ABA, although they might also play a role in plant response to biotic stress imposed by necrotrophic pathogens and regulated by JA-dependent pathways.…”

“…In addition to their key structural function, sterols also play essential roles in modulating plant growth and development, not only because campesterol is the biosynthetic precursor of the brassinosteroid hormones (Yokota, 1997) but also because changes in sterol composition directly affect a number of cell processes, such as vascular and stomatal patterning (Jang et al, 2000; Carland et al, 2002; Qian et al, 2013), cell division, expansion and polarity (He et al, 2003; Men et al, 2008), cell-to-cell connectivity (Grison et al, 2015), hormonal regulation (Souter et al, 2002; Kim et al, 2010), vacuole trafficking (Li et al, 2015), cell wall formation (Schrick et al, 2012), pollen viability (Ischebeck, 2016) and even proper plastid development (Babiychuk et al, 2008; Kim et al, 2010; Itkin et al, 2012; Manzano et al, 2016). The specific contribution of glycosylated sterols, particularly of SG, to these processes is far from being fully understood, although there is increasing evidence supporting an important role of the ratio of conjugated to free sterol forms in regulating the properties of the cell membranes (Moreau et al, 2002; Grille et al, 2010; Grosjean et al, 2015) and therefore of different PM-associated processes like plant adaptation to biotic and abiotic stress conditions (Lynch and Steponkus, 1987; Palta et al, 1993; Uemura and Steponkus, 1994; Moreau et al, 2002; Minami et al, 2009; Mishra et al, 2013; Li et al, 2014; Pandey et al, 2014; Tarazona et al, 2015; Saema et al, 2016; Singh et al, 2016; Takahashi et al, 2016), signaling and transport, and recruitment of proteins to specific membrane subcompartments (Zauber et al, 2014). SGs have also been suggested to serve as primers for ceramide glycosylation (Lynch et al, 1997) and cellulose biosynthesis (Peng et al, 2002; Li et al, 2014), but whether SGs are primers for cellulose synthesis in vivo still remains an open question (Schrick et al, 2012).…”

Identification and functional characterization of DzS3GT, a cytoplasmic glycosyltransferase catalyzing biosynthesis of diosgenin 3-O-glucoside in Dioscorea zingiberensis

“…Four full-length SGTs have been cloned using rapid amplification of cDNA ends (RACE) method. The expression of SGT genes was significantly up-regulated after methyl jasmonate or salicylic treatment indicating their possible roles in defense mechanisms (Saema et al 2016).…”