samples, and separated by two-dimensional gel electrophoresis (2D-GE). This form of
electrophoresis separates proteins based on their isoelectric point in the first dimension,
and on their molecular weight in the second dimension. After electrophoresis the gels
were autoradiographed, leading to protein-spot profiles for the extracts under investiga-
tion. When protein profiles from control and gravistimulated seedlings were compared, 2
out of approximately 600 detectable spots increased in intensity in the gravistimulated
samples relative to control. A similar experiment testing the effect of continuous stimu-
lation by rocking the dishes over a period of 24 hours led to the identification of 10 grav-
ity up-regulated and 4 gravistimulation-specific protein spots (Sakamoto et al. 1993).
To detect potential differences in protein phosphorylation upon stimulation, control
and continuously rocked samples were labeled with^32 P-orthophosphate during the stim-
ulus. Proteins that were phosphorylated during the period of treatment should appear as
radioactive spots on the 2D-GE gels. By comparing radioactive protein spots between
control and continuously rocked samples, Sakamoto et al. (1993) were able to demon-
strate that continuous rocking enhances the phosphorylation of two protein spots. Hence,
an important conclusion of these studies is that gravity- and/or mechano-stimulation pro-
mote changes in 2D-GE protein spot intensity, reflective of changes in protein abundance
and/or post-translational modification, and differential phosphorylation of specific pro-
teins in Arabidopsis thalianaseedlings.
Another attempt at establishing a role for protein phosphorylation in gravity signal
transduction sought phosphoproteins with differential levels of expression between upper
and lower flanks of gravistimulated oat pulvini (Chang and Kaufman 2000; Chang et al.
2003). These investigations uncovered two soluble and two membrane-associated pro-
teins that are differentially phosphorylated in lower versus upper pulvinus halves in re-
sponse to gravistimulation. Subsequent work defined more thoroughly the gravity-
induced phosphorylation of one of the soluble oat proteins, demonstrating that it occurs
as early as 5 min after initiation of gravistimulation and requires a newly synthesized pro-
tein. This time of initial phosphorylation correlates well with the minimal gravistimula-
tion time needed to activate a productive transduction pathway leading to curvature re-
sponse (presentation time), which is 5.2 min in oat pulvini. The differentially
phosphorylated 50kD oat protein is itself a kinase, as demonstrated in autophosphoryla-
tion experiments. Altogether, these data indicate that the differential phosphorylation of
this 50kD protein in graviresponding oat pulvini might contribute to gravity signal trans-
duction in this system (Chang et al. 2003).
In these early proteomic experiments, no attempts were made to identify the proteins
present in the differentially represented 2D-GE protein spots (Sakamoto et al. 1993;
Chang et al. 2003). However, the last decade witnessed amazing developments in mass
spectrometry that truly revolutionized our ability to identify proteins based on their mass,
on the mass of their proteolytic products, and on their amino acid content (Li and
Assmann 2000). Taking advantage of this technological revolution, researchers are now
able to identify differentially represented proteins as long as they are working with an or-
ganism whose genome has been completely sequenced. It is not surprising that recent
proteomic studies have identified a number of Arabidopsisproteins whose abundance or
modification varies in response to gravistimulation.
First, Kamada et al. (2005) identified proteins associated with the cytoskeleton and the
frankie
(Frankie)
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