Agriculture Reference
In-Depth Information
and functional in most crop species; (iv) inexpensive and capable of being used
everywhere; (v) simply to detect with little or no instrumentation; (vi) easy to use
under field conditions.
Naturally fluorescent proteins could offer a valuable alternative to the use of
GUS reporter systems since, in contrast to GUS, the detection of their expression
does not require the addition of a substrate. Green fluorescent protein (GFP), a
spontaneously fluorescent protein, was initially isolated from the luminescent
marine jellyfish ( Aequorea victoria ). GFP emits a highly and stable bright green
fluorescence after absorbing blue light (Tsien 1998 ). The wild type Aequorea
protein has a major excitation peak at 395 nm which is about three times higher
in amplitude than a minor peak at 475 nm. In normal solution, excitation at 395 nm
gives emissions peaking at 508 nm, whereas excitation at 475 nm gives a maximum
at 503 nm (Heim et al. 1994 ). Since its discovery GFP from Aequorea victoria has
become a frequently used tool in plant biology. The first studies on transgenic plants
expressing wild type GFP proved the usefulness of this protein as an in vivo and
real-time visible marker and encouraged researchers to modify it in order to obtain
new variants that could be more effectively synthesised in plant cells and macro-
scopically detectable at the whole plant level (Stewart 2001 ). One of these modified
versions of GFP is the mGFP5er variant that produces a stable protein targeted to
the endoplasmic reticulum as the result of the addition of a N-terminal Arabidopsis
basic chitinase fusion and a C-terminal HDEL fusion (Haseloff et al. 1997 ). The
coding sequence contains three mutations that enhance the folding of mGFP5er at
higher temperatures and allows excitation of the protein using either ultraviolet
(395 nm) or blue (473 nm) light (Siemering et al. 1996 ). In addition, new fluores-
cence colours have been created through mutagenesis of the natural protein giving
longer excitation and emission wavelengths and to enhance the fluorescence bright-
ness. The new colours range from blue and cyan (EBFP and ECFP) to yellow
(EYFP); such new proteins have excitation/emission peaks at 383/474, 434/472 and
514/527 nm, respectively (Spiess et al. 2005 ; Mena et al. 2006 ).
Fluorescent proteins have been largely used as visual genetic labels at the whole
plant, tissue and cell levels, since they offer a fast and easy-to-use non-destructive
tool with which the efficiency and timing of gene expression can be evaluated.
Detection and quantification of fluorescence at the whole-plant level normally
requires the use of complex and expensive laboratory instruments (scanning laser
and fluorescence imaging systems). Portable instruments, such as fibre optic probe
fluorometers, have recently been designed to assess GFP fluorescence under field
conditions (Harper and Stewart 2000 ; Millwood et al. 2003 ). However, because
bioindicators need to be disseminated over a wide area, a successful field applica-
tion of these plants also requires a cost-effective remote monitoring system pro-
viding real-time information about the nutritional status of a whole crop system.
Recently Adams et al. ( 2011 ) proposed an alternative method for crop monitoring
in which sentinel plants and sensing units are deployed in tandem at specific
locations. Ideally, such a system integrates biological and sensory technologies
with communication technologies to provide a practical field-deployable telemetry
system.
Search WWH ::




Custom Search