Time-Lapse Imaging of Developing Meristems Using Confocal Laser Scanning Microscope - Plant Cell Morphogenesis: Methods and Protocols - page 109

the meristem [ 1 ]. The genetic basis of meristem function was then
analyzed: many mutants with an altered number of organs or with
defective organ identities were isolated from the 1980s onwards
(e.g., ref. [ 2 ]). The observation of mutant meristems was con-
ducted, and in parallel, gene expression was monitored with
reporter gene expression (e.g., promoter activity with GUS activ-
ity), in situ hybridization (mRNA accumulation in meristem sec-
tions), or immunolocalization (e.g., ref. [ 3 ]). The impact of
mutations on meristem shape was analyzed, using in particular
scanning electron microscopy (e.g., ref. [ 4 ]). The generalization of
the use of confocal laser scanning microscopes (CLSM) in the
1990s allowed a more precise analysis of cell behavior and meri-
stem shape in 3D while being still mostly limited to fi xed tissues
(e.g., ref. [ 5 ]). The localization of certain GFP-fused proteins in
the meristem was initiated, notably to show the existence of trans-
port mechanisms across meristem layers (e.g., ref. [ 6 ]). In 2004,
two studies were published, in which whole meristems were
observed over time [ 7 , 8 ]. Today, this switch to live imaging still
represents a major step forward in the analysis of meristem biology
and this is what this protocol paper is about.
Two plant model systems have received more attention for
time-lapse imaging, namely, the inflorescence meristem of
Arabidopsis thaliana and the vegetative meristem of Lycopersicon
esculentum (tomato). As meristems are usually covered by young
organs, this hinders their analysis under a microscope. Young
organs are thus dissected out, and cut stems can be grown in vitro
[ 7 , 9 - 12 ]. Alternatively, whole plantlets can be grown in vitro in
the presence of the auxin transport inhibitor (and fl ower formation
inhibitor) NPA to generate naked meristems [ 8 , 13 ]. Meristems
can be observed with a CLSM equipped with a water-dipping lens
in water at room temperature over time, which makes them ideal
systems to study the cellular basis of morphogenesis in the aerial
part of the plant. In particular, using transgenic lines, one can fol-
low gene expression or protein dynamics, such as the polarity of
the auxin effl ux carrier PIN1 or cortical microtubule orientations,
and associate these behaviors with shape changes in the developing
meristems (e.g., refs. [ 14 , 15 ]). Last, these living meristems can be
treated with drugs—e.g., the microtubule-depolymerizing drug
oryzalin [ 8 , 16 ]—and hormones, e.g., auxin [ 17 ], and the tissue
can be mechanically perturbed by local compressions or laser-
induced cell ablations [ 13 , 18 ]. Other imaging techniques can be
used sequentially to obtain additional information on the meri-
stems, such as high-resolution growth quantifi cations with the rep-
lica method [ 19 - 21 ] or with other 3D reconstruction methods
such as MARS-ALT [ 12 ] or MorphoGraphX [ 22 ], as well as local
mechanical properties with indentation techniques [ 23 , 24 ].