Quantifying Fluorescent Protein Expression

The scanning fluorescence
spectroscopy apparatus (Fluorolog-3, Jobin Yvon and Glen Spectra,
Edison, NJ) equipped with the FluorEssence Software (HORIBA Scientific,
version 3.8.0.60) was used to quantify the fluorometric signal from
leaf tissue as described previously.^{46 (link)} The
GFP, RFP, and BFP signals were analyzed at excitation wavelengths
of 475, 550, and 400 nm, and emission ranges of 509, 574, and 455
nm, respectively, to collect the maximum emission peaks. Six measurements
per infiltrated plant were collected from the same leaf tissue used
for protoplasts isolation. A minimum of three independent plants per
construct was analyzed. Fluorometric data was processed using Microsoft
Excel software as previously described.^{46 (link)} The results are expressed as mean ± standard deviation of log₁₀ of CPS (counts per second). To ensure that detected fluorescence
was not the direct result of A. tumefaciens, cultures containing constructs with promoters and 5′ UTRs
from A. tumefaciens were checked for
expression on a plate reader (Figure S2).