TITLE:
Tissue Specific Expression of a Terpene Synthase in Nicotiana benthamiana Leaves
AUTHORS:
Seifu Juneidi, Hieng Ming Ting, Alexsander van der Krol
KEYWORDS:
Cell Type Specific Expression, GUS, Linalool Synthase (FaNES1), Promoter
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.5 No.18,
August
28,
2014
ABSTRACT:
To study transient tissue specific linalool emission and to examine its
fate, plastid targeted dual linalool/nerolidol synthase (FaNES1) was introduced
into Nicotiana benthamiana leaves
under LTP1, SUC2, RbcS and CaMV35 promoters. Tissue specificity of the
promoters was tested with β-glucuronidase
(GUS) reporter gene. Promoter::GUS/FaNES1 fusion was confirmed with colony PCR
and agro-infiltrated into six weeks old N.
benthamiana leaves. Eight days post inoculation (dpi), promoter::GUS
constructs were examined with GUS histochemical staining at 1.5 h, 3.5 h, 5.5 h
and 16 h incubation times. After 4/8 days, FaNES1 construct agro-inoculated
leaves were assessed for linalool emissions and its conjugates using a dynamic
headspace system and LC-MS, respectively. There was high affinity of promoters
to their respective cell-types although it was not as specific as in stable
transformation. This is possibly due to activations of many copies of transiently
introduced promotes by few transcription factors of the respective promoter
that naturally exist in untargeted cell-types. GUS staining intensity was
stronger in leaf veins and injured sites compared to other plant tissues under
all heterologous promoters, and it was gradually increased with increase in
incubation times that could be explained by promoters wounding responses and/ or
GUS leakage to unstained sites. Linalool emission was obtained in the same
pattern under all promoters; it was higher at day 4 than day 8. Its
concentration declined 44, 12, 4.5 and 4 folds at 8 dpi under LTP1, SUC2, RbcS
and CaMV35S promoters, respectively. Conversely, linalool conjugates were
significantly increased at day 8. These might be due to T-DNA degradations
and/or protein modifications 4 dpi. LTP1 promoter was the least efficient to
drive both GUS and FaNES1 possibly due to immature plastids in epidermal cells
and/or its weak performance. Hence, to study FaNES1 activity in transient assay
it is suggested to use relatively shorter duration and longer inoculation times
for linalool and its conjugates, respectively.