such localized fluxes remain the reason no Ca2+signal has been localized to the gravisens-
ing cells of the plant. Alternatively, the circumstantial evidence for Ca2+signaling may be
misleading and gravisignaling may reside in some other transduction pathway.
Also as a note of caution, the coelentrazine cofactor for aequorin required to reconsti-
tute the active Ca2+sensor is itself exquisitely sensitive to ROS. In response to ROS it
generates a signal similar to that expected of an increase in Ca2+(Lucas and Solano 1992;
Plieth 2005; Molecular Probes 2006). Therefore, experiments using this approach require
careful controls for possibly confounding effects of ROS production. Such controls are
especially important as there are reports that transient changes in ROS production are as-
sociated with the auxin fluxes generated during the graviresponse (Joo et al. 2001; Joo et
al. 2005).
Similarly, sustained mechanical stress induces ROS production in suspension-cultured
soybean and parsley cells within 4 to 10 min (Yahraus et al. 1995; Gus-Mayer et al. 1998),
suggesting that ROS may also complicate aequorin-based measurements involving me-
chanical stimulation. ROS are now thought to participate in many plant response systems
(Mori and Schroeder 2004), and the finding that they in turn affect ROS-gated Ca2+chan-
nels (Pei et al. 2000; Demidchik et al. 2003; Foreman et al. 2003) makes them strong can-
didates for regulators in mechano-/graviperception. To make the story even more com-
plex, ROS may well be involved both at the level of intracellular signaling elements (Mori
and Schroeder 2004) and also as factors modulating cell wall properties associated with
growth (Campbell and Sederoff 1996; Brady and Fry 1997; Coelho et al. 2002; Kerr and
Fry 2004).
5.5 Insights from transcriptional profiling
The initial identification of touch-responsive (TCH) genes in Arabidopsiswas achieved
through differential cDNA screening of plants stimulated by touch or wind (Braam and
Davis 1990). The identity of many of these genes as encoding Ca2+-binding proteins re-
inforced the theme of Ca2+-dependent signaling in the mechanical response of the plant.
Thus,TCH1encodes CaM2 (one of the ArabidopsisCaMs) and TCH2andTCH3encode
CML24 and CML12, both CaM-like proteins (Braam and Davis 1990; Sistrunk et al.
1994; Khan et al. 1997; McCormack and Braam 2003). Similar analysis has now identi-
fied a range of genes showing mechanosensitive expression, including other CaMs (Ling
et al. 1991; Perera and Zielinski 1992; Gawienowski et al. 1993; Botella and Arteca 1994;
Ito et al. 1995; Botella et al. 1996; Oh et al. 1996).
However, the expression levels of many non-Ca2+-related genes have also been shown
to be touch-responsive. For example, TCH4codes for a cell wall-modifying enzyme, a
xyloglucan endotransglucosylase/hydrolase (Xu et al. 1995). Indeed, the genome-wide
view afforded by microarray transcript profiling has revealed a remarkably rapid and
widespread alteration in the spectrum of mRNA present after both touch and gravistim-
ulation. For example, Kimbrough et al. (2004) reported approximately 1,700 transcripts
changing in abundance after either mechanical or gravity stimulation of Arabidopsis
roots, representing approximately 7% of the genome. Many of the transcriptional changes
were found to be common to both touch and gravistimulation, perhaps reflecting the sim-