Ectopic overexpression of peach GDP-d-mannose pyrophosphorylase and GDP-d-mannose-3′,5′-epimerase in transgenic tobacco

Abstract

We created transgenic tobacco overexpressing peach GDP-D-mannose pyrophosphorylase (PpGMPH) and GDP-D-mannose-3 0 ,5 0 -epimerase (PpGME), which are involved in the main L-ascorbate (Asc) biosynthetic pathway in plants. Despite an apparent increase in enzymatic activity in the PpGMPH-overexpressing primary transgenic (T0) lines, no significant increase in Asc pool size was observed in leaves. For PpGME-overexpressing T0 lines, Asc pool size was also unchanged, although transcriptional overexpression was confirmed. To reinforce the biosynthetic pathway from D-mannose-1-phosphate to GDP-L-galactose, we further created PpGMPH-and PpGME-double overexpressing crossed transgenic (dOx T1) lines. Transcriptional overexpression of PpGMPH and PpGME transgenes was confirmed, and the Asc content in the dOx T1 lines was slightly elevated in young leaves, albeit not significantly. Western blot analyses revealed protein amounts of GMPH and GME were similar among dOx T1, parental T0 and non-transgenic (SR1) lines in three tissue types tested: leaves, flower buds and immature fruits. Moreover, a similar amount of GMPH and GME proteins were detected in young leaves (YL) and old leaves (OL), although Asc content in YL is roughly double as that in OL. In vitro assay using recombinant PpGME suggested the equilibrium of GME reaction is unfavorable to forward Asc biosynthesis at this step. Finally, to investigate substrate availability in relation to Asc pool size, we added exogenous GDP-D-mannose (5 mM) or L-galactose (5 mM) to tobacco leaf discs; the former resulted in no increase in Asc content, whereas a significant increase was observed in the latter, not only in the dOx T1 lines, but also in the original line SR1. Similar treatments were also conducted under high light, which resulted in a further increase in the increment of Asc content in the L-galactose-fed discs irrespective of transgene expression level. Taken together, both L-galactose availability and light rather than upper biosynthetic gene expression levels of GMPH/GME are critical determinant of Asc content in tobacco leaves, indicating complex modulation of this compound.Keywords L-ascorbate Á GDP-D-mannose-3 0 ,5 0 -epimerase Á GDP-D-mannose pyrophosphorylase Á Overexpression Á Transgenic tobacco Abbreviations Asc L-Ascorbate

Manipulation of Ascorbate Biosynthetic, Recycling, and Regulatory Pathways for Improved Abiotic Stress Tolerance in Plants

“…Increased co-expression of the GMP + GME + GGP + GPP genes in tomato also increased ascorbate concentrations 2.0-fold; however, this was the same fold-change for increased expression of the GGP gene alone, suggesting that GMP is not a major rate-limiting step in the L-galactose pathway in tomato [26]. This conclusion is also supported by other studies increasing the expression of the GMP gene in Arabidopsis and tobacco and increasing the co-expression of the GMP + GME genes in tobacco that did not report significant changes in ascorbate concentrations [29,30] (Table 1).…”

“…Increased expression of the GMP + GME + GGP + GPP genes in tomato only increased ascorbate concentrations 2.0-fold, the same fold-change for increased expression of the GGP gene alone, suggesting that GME is not a major rate-limiting step in the L-galactose pathway in tomato [26]. Moreover, increased expression of the GME gene in tobacco did not significantly change ascorbate concentrations, suggesting the GME is not a rate-limiting step of the L-galactose pathway in tobacco [29]. Increased expression of the GME gene in plants has been associated with enhanced tolerance to salt, low pH, drought, cold, and methyl viologen-induced stress [28,33,35] (Table 1).…”

Manipulation of the Rice L-Galactose Pathway: Evaluation of the Effects of Transgene Overexpression on Ascorbate Accumulation and Abiotic Stress Tolerance

“…To date, four alternative AsA biosynthesis pathways have been proposed in plants: the L-galactose [ 9 ], galacturonate [ 10 , 11 ], glucose [ 12 ] and myo-inositol pathways [ 13 ]. Among these pathways, the L-galactose pathway might have an important role in cereal AsA biosynthesis ( Fig 1 ) [ 14 ]. For example, when rice seedlings were supplied with L-galactose and L-galactono-1,4-lactone, shoot AsA contents increased up to 5- and 4-fold, respectively; in contrast, for D-galacturonic acid, myo-inositol, D-glucuronolactone and L-guluno-1,4-lactone, AsA levels were similar to those of the untreated control plants [ 15 ].…”

Medicine is not health care, food is health care: plant metabolic engineering, diet and human health

“…Enhancement strategies have focused on increasing the activity of key biosynthetic enzymes, and elevation of the activity of GGP has had the greatest effect on concentrations of vitamin C giving 6.2-fold more in tomato fruit (up to 111 mg g À1 FW), 3.0fold more in potato tubers (up to 36 mg g À1 FW), 2.1-fold more in strawberries (up to 131 mg g À1 FW) (Bulley et al, 2012) and 2.5fold more in rice (Zhang et al, 2015a). Combined expression of GGP/vtc2 and the gene encoding GDP-mannose epimerase (GME/ vtc4) has been attempted to overcome the problem of GME becoming rate-limiting in plants with high GGP activity, but without very strong effects on ascorbate concentrations in leaves (Imai et al, 2012;Macknight et al, 2017). Recent results showing post-transcriptional regulation of GGP production via an untranslated open reading frame (ORF) in the 5 0 untranslated region of the GGP/vtc2 gene in response to ascorbate concentrations, suggest that removal of this homeostatic regulatory unit (by mutation or genome editing) could elevate ascorbate synthesis substantially (Laing et al, 2015).…”