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′,5′-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.