TITLE:
Functional Aspects of Silencing and Transient Expression of psbS in Nicotiana benthamiana
AUTHORS:
Richard B. Peterson, Hillar Eichelmann, Vello Oja, Agu Laisk, Eero Talts, Neil P. Schultes
KEYWORDS:
Fluorescence; Gas Exchange; Nonphotochemical Quenching; Quantum Yield; 810-nm Absorbance
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.4 No.7,
July
17,
2013
ABSTRACT: MicroRNA-based
gene silencing is a functional genomics tool for a wide range of eukaryotes. As
a basis for broader application of virus-induced gene silencing (VIGS) to
photosynthesis research, we employed a tobacco rattle virus (TRV) vector to
silence expression of the nuclear psbS gene in Nicotiana benthamiana. The
22-kiloDalton psbS protein is essential for xanthophyll- and H+-dependent
thermal dissipation of excitation in higher plants widely known as nonphotochemical
quenching (NPQ). Controls treated with the TRV-VIGS vector containing a
bacterial chloramphenicol resistance gene as the silencing target were included
to test for non-silencing effects of the viral vector system. PsbS protein was
undetectable and both psbS mRNA
transcript levels and NPQ capacity were dramatically reduced in new leaf tissue
of VIGS-psbS plants only. Photosynthetic
performance in TRV-VIGS-treated and uninfiltrated plants was assessed by
application of CO2 exchange, chlorophyll fluorescence, and in vivo absorbance changes at 810 nm. TRV-VIGS caused a
mild stress based on pigment content and light absorption characteristics in
some cases. To assess transient complementation of NPQ, the endogenous psbS gene was silenced using only the
transit sequence in the TRV vector followed by Agrobacterium-mediated transient
expression of a modified gene consisting of an altered transit sequence fused
to the native mature protein sequence. Nevertheless, NPQ in infused fully
expanded leaves that expressed this re-introduced form was not fully restored
indicating the possible importance of psbS incorporation prior to formation of
grana stacks.