Spatial and temporal regulation of sterol biosynthesis in Nicotiana benthamiana

Abstract

Nicotiana benthamiana was used as a model to investigate the spatial and developmental relationship between sterol synthesis rates and sterol content in plants. Stigmasterol levels were approximately twice the level in roots as that found in aerial tissues, while its progenitor sterol sitosterol was the inverse. When incorporation of radiolabeled precursors into sterols was used as measure of in vivo synthesis rates, acetate incorporation was similar across all tissue types, but approximately twofold greater in roots than any other tissue. In contrast, mevalonate incorporation exhibited the greatest differential with the rate of incorporation in roots approximately one-tenth that in apical shoots. Similar to acetate, incorporation of farnesol was higher in roots but remained fairly constant in aerial tissues, suggesting less regulation of the downstream sterol biosynthetic steps. Consistent with the precursor incorporation data, analysis of gene transcript and measurements of putative rate-limiting enzyme activities for 3-hydroxy-3-methylglutaryl-coenzyme A synthase (EC 2.3.3.10) and reductase (EC 1.1.1.34) showed the greatest modulation of levels, while the activity levels for isopentenyl diphosphate isomerase (EC 5.3.3.2) and prenyltransferases (EC 2.5.1.10 and EC 2.5.1.1) also exhibited a strong but moderate correlation with the development age of the aerial tissues of the plants. Overall, the data suggest a multitude of means from transcriptional to posttranslational control affecting sterol biosynthesis and accumulation across an entire plant, and point to some particular control points that might be manipulated using molecular genetic approaches to better probe the role of sterols in plant growth and development.

Why Do Plants Convert Sitosterol to Stigmasterol?

“…Similar to the findings of Kemp et al. (1967), N. benthamiana seedlings display striking differences in sterol composition between organs, with higher stigmasterol content in roots than in leaves (Suza and Chappell, 2016). In contrast, stigmasterol concentration is elevated in P. sativum leaves but lower in seeds (Schrick et al., 2011).…”

“…In mammalian systems, cholesterol biosynthesis is regulated via negative feedback suppression of several key genes (Goldstein and Brown, 1990). Thus, analysis of gene transcripts may help increase our understanding of the regulation of sterol biosynthesis during plant development (Schrick et al., 2011; Sonawane et al., 2016; Suza and Chappell, 2016). For instance, in the developing seeds of tobacco ( N. tabacum ), pea ( Pisum sativum ), rape ( Brassica napus ), and seedlings of N. benthamiana and B. napus , increased gene expression and enzyme activities coincides with sterol accumulation (Harker et al., 2003; Schrick et al., 2011; Suza and Chappell, 2016).…”

“…Thus, analysis of gene transcripts may help increase our understanding of the regulation of sterol biosynthesis during plant development (Schrick et al., 2011; Sonawane et al., 2016; Suza and Chappell, 2016). For instance, in the developing seeds of tobacco ( N. tabacum ), pea ( Pisum sativum ), rape ( Brassica napus ), and seedlings of N. benthamiana and B. napus , increased gene expression and enzyme activities coincides with sterol accumulation (Harker et al., 2003; Schrick et al., 2011; Suza and Chappell, 2016). In addition, apical tissues of B. campestris contain high levels of cholesterol but exhibit a decline in cholesterol and a rise in sitosterol at later stages of development (Hobbs et al., 1996).…”

Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants

“…For instance, such method enabled the elucidation of podophyllotoxin biosynthesis in Podophyllum hexandrum 22 . Furthermore, N. benthamiana accumulates substantial amount of cholesterol (∼ 12% of total sterols) 23 , and thus is well suited for screening candidate enzymes for diosgenin biosynthesis. We reasoned that if the minimum set of diosgenin-biosynthetic CYP s were contained within a larger test gene pool, co-expression of this pool of genes in N. benthamiana would likely result in diosgenin production, and the specific CYP s could be subsequently identified.…”

Metabolic networks of the Nicotiana genus in the spotlight: content, progress and outlook

“…As a consequence, the pathway was well characterized in animals but poorly understood in plants [ 68 ]. However, solanaceous plants can accumulate cholesterol to significantly higher levels [ 69 ] and may reach up to 12% of total sterols in N. benthamiana [ 70 ]. It was not until 2014 that the first committed enzyme involved in the biosynthesis of cholesterol in plants was characterized.…”