The bHLH Transcription Factors TSAR1 and TSAR2 Regulate Triterpene Saponin Biosynthesis in Medicago truncatula

Abstract

Plants respond to stresses by producing a broad spectrum of bioactive specialized metabolites. Hormonal elicitors, such as jasmonates, trigger a complex signaling circuit leading to the concerted activation of specific metabolic pathways. However, for many specialized metabolic pathways, the transcription factors involved remain unknown. Here, we report on two homologous jasmonate-inducible transcription factors of the basic helix-loop-helix family, TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR1 (TSAR1) and TSAR2, which direct triterpene saponin biosynthesis in Medicago truncatula. TSAR1 and TSAR2 are coregulated with and transactivate the genes encoding 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE1 (HMGR1) and MAKIBISHI1, the rate-limiting enzyme for triterpene biosynthesis and an E3 ubiquitin ligase that controls HMGR1 levels, respectively. Transactivation is mediated by direct binding of TSARs to the N-box in the promoter of HMGR1. In transient expression assays in tobacco (Nicotiana tabacum) protoplasts, TSAR1 and TSAR2 exhibit different patterns of transactivation of downstream triterpene saponin biosynthetic genes, hinting at distinct functionalities within the regulation of the pathway. Correspondingly, overexpression of TSAR1 or TSAR2 in M. truncatula hairy roots resulted in elevated transcript levels of known triterpene saponin biosynthetic genes and strongly increased the accumulation of triterpene saponins. TSAR2 overexpression specifically boosted hemolytic saponin biosynthesis, whereas TSAR1 overexpression primarily stimulated nonhemolytic soyasaponin biosynthesis. Both TSARs also activated all genes of the precursor mevalonate pathway but did not affect sterol biosynthetic genes, pointing to their specific role as regulators of specialized triterpene metabolism in M. truncatula.

The genome of Chenopodium quinoa

“…Specifically, AUR62017204 and AUR62017206 are neighbouring genes annotated as basic helix-loop-helix (bHLH) transcription factors sharing homology (Extended Data Fig. 9a) with the class IVa bHLH genes that are known to regulate triterpenoid biosynthesis in Medicago trancatula 39 . In M. truncatula, overexpression of the triterpene saponin biosynthesis activating regulator 1 (TSAR1) and TSAR2 bHLH transcription factors was recently shown to increase the expression of genes in the triterpenoid biosynthetic pathway, resulting in increased accumulation of triterpene saponins 39 .…”

“…9a) with the class IVa bHLH genes that are known to regulate triterpenoid biosynthesis in Medicago trancatula 39 . In M. truncatula, overexpression of the triterpene saponin biosynthesis activating regulator 1 (TSAR1) and TSAR2 bHLH transcription factors was recently shown to increase the expression of genes in the triterpenoid biosynthetic pathway, resulting in increased accumulation of triterpene saponins 39 . TSAR1 and TSAR2 were also found to bind to the DNA motif 5′ -CACGHG-3′ (where H can be A, C, or T) 39 .…”

“…In M. truncatula, overexpression of the triterpene saponin biosynthesis activating regulator 1 (TSAR1) and TSAR2 bHLH transcription factors was recently shown to increase the expression of genes in the triterpenoid biosynthetic pathway, resulting in increased accumulation of triterpene saponins 39 . TSAR1 and TSAR2 were also found to bind to the DNA motif 5′ -CACGHG-3′ (where H can be A, C, or T) 39 . In quinoa, we found that AUR62017206 (hereafter TSAR-like 2, TSARL2) was expressed in root tissue but not in flowers or immature seeds, whereas AUR62017204 (hereafter TSARL1) was almost exclusively expressed in seeds, with significantly lower expression levels in sweet lines (Supplementary Data 8).…”

“…The alignment of bHLH domains was performed with Clustal Omega 63 , using sequences from Mertens et al 39 . The phylogeny was inferred using the maximum likelihood method based on the JTT matrix-based model 67 .…”

Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids

“…Further studies, such as analyses during the developmental stages of russeted vs waxy‐skinned apple cultivars, would be needed to identify transcription factors that are involved in the fine regulation of apple triterpenic acids. Interestingly, a recent study (Mertens et al ., ) reported the identification of two homologous transcription factors that distinctly direct triterpene saponin biosynthesis in M. trunculata .…”