Biology Reference
In-Depth Information
thaliana
, our mechanistic understanding of how the microtubule cytoskeleton affects
plant life has dramatically increased. It is a simple process to construct transgenic
A. thaliana
plants that express fluorescent protein fusions by using the disarmed
plant pathogen
Agrobacterium tumefaciens
. Several screening steps are necessary
to ensure that the fusion protein accurately mimics the native protein because trans-
genes are inserted randomly into the
A. thaliana
genome. To image the fluorescent
proteins
in planta
, confocal microscopy is used to alleviate issues caused by speci-
men thickness and autofluorescence.
INTRODUCTION
Plants are essential for human livelihood because they produce food, fuel, shelter, and
many medicines. Because plant products are important to our society, there is much in-
terest in understanding the complex mechanisms that govern plant growth and devel-
opment in different environmental contexts. Several lines of evidence indicate that
the microtubule cytoskeleton is a key component of the growth machinery in all organ-
isms, including plants (
Chan, 2012; Lloyd, 2011; Lucas & Shaw, 2008; Nick, 2012;
Wasteneys, 2004
). However, we know relatively little about the molecular relationship
between plant growth and the microtubule cytoskeleton (
Crowell, Gonneau, Stierhof,
Hofte, & Vernhettes, 2010; Smith & Oppenheimer, 2005; Szymanski & Cosgrove,
2009;Wasteneys&Fujita, 2006
). The ability to label and image proteins of interest with
fluorescent proteins in living cells has allowed researchers to build a deeper mechanistic
understanding of biology. In this review, wewill focus on techniques used to image fluo-
rescent protein reporters in living seedlings of
Arabidopsis thaliana. A. thaliana
is a
model flowering plant for genetics, development, and cell biology. Specifically, we will
discuss imaging microtubules and microtubule-associated proteins (MAPs).
In most plant cells, four types of microtubule arrays cyclically appear throughout
the cell cycle: the interphase cortical array, the preprophase band, the spindle, and the
cytokinetic phragmoplast (
Muller, Wright, & Smith, 2009; Van Damme & Geelen,
2008
). Genetic or chemical disruption of any array severely disrupts growth and mor-
phogenesis (
Baskin, Wilson, Cork, & Williamson, 1994; Corson et al., 2009
). As in
other organisms, the spindle segregates genetic material duringM-phase. The prepro-
phase band and phragmoplast are unique to plants, and both are required for the ac-
curate positioning of new cell walls (
Rasmussen, Humphries, & Smith, 2011; Smith,
2001
). The precise placement of new cell walls is an important patterning step in plant
development, and the mechanisms in which the preprophase band and phragmoplast
mediate this process are largely unknown (
De Smet &Beeckman, 2011; Muller et al.,
2009; Van Damme, Vanstraelen, & Geelen, 2007
). The microtubules of the prepro-
phase band and interphase array are linked along their lengths to the plasmamembrane
by currently unknown proteins (
Hardham&Gunning, 1978
). The interphase array af-
fects properties of the cellulosic cell wall, which affects cell expansion and therefore
growth and morphology (
Chan, 2012; Lloyd, 2011; Lucas &Shaw, 2008; Nick, 2012;
Wasteneys, 2004
). Depending upon environmental conditions and the developmental
context, interphase arrays are organized into different configurations (
Fig. 15.1
;
