References

408 L.G. Perry et al.
phytotoxins in C. maculosa root exudates needed to be identified to evaluate
the role of allelopathy in C. maculosa dominance over North American
grassland species.
27.3.1
Identification of the Allelochemical
To overcome the difficulty of isolating root-secreted allelochemicals from
soil, Bais et al. (2002) grew C. maculosa in vitro and collected the root
exudates in sterile media. Addition of crude C. maculosa exudates to liquid
media where Linaria dalmatica (Dalmatian toadflax), Verbascumthapsus
(common mullein), Bromus tectorum (downy brome), Kochia scoparia
(kochia), C. diffusa (diffuse knapweed), and Arabidopsis thaliana were
growing reduced root growth and induced plant mortality by 14 days
after treatment, indicating that a component of the root exudates had
broad-spectrum phytotoxic activity (Bais et al. 2002). The active fraction of
the exudates was identified as (±)-catechin using high-performance liquid
chromatography separations and identification by mass spectrometry and
1 Hand 13 C NMR techniques. By testing commercially purchased isomers of
pure catechin, Bais et al. (2002) initially attributed the phytotoxic activity
to (–)-catechin. However, it was recently determined that (+)-catechin is
also phytotoxic (Iqbal et al. 2003), although less potent than (–)-catechin
(Veluri et al. 2004a).
27.3.2
Catechin Induces Reactive Oxygen Species
and Ca 2+ -Mediated Cell Death
As is the case for many allelochemicals, the exact cellular target of (±)catechin
is still unknown. However, many of the cell signaling and indirect
molecular events that precede catechin-mediated cell death have been identified.
Using real-time video imaging and fluorescent viability dyes, with
A. thaliana and C. diffusa as target species, Bais et al. (2003) observed
that cell death began about 15 min after catechin treatment, following condensation
of the cytoplasm. Cell death occurred first in the meristematic
zone of the root tip and moved upward to the central elongation zone, suggesting
that catechin or catechin-induced signals are transported through
the vascular system. To more fully understand the molecular basis for
catechin-mediated cell death, Bais et al. (2003) monitored the kinetics of
several cellular signals, including generation of reactive oxygen species
(ROS), fluctuations in cytoplasmic calcium concentrations ([Ca 2+ ]cyt), and