Agriculture Reference
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centration of cytoplasmic calcium ions ([Ca 2+ ] cyt ). Not surprisingly, then,
scientists interested in testing the signaling roles of eATP and extracellular
ADP(eADP)inplantsassayedtheeffectsofthesenucleotidesonmembrane
transport properties.
The first report of the rapid induction of membrane potential changes
by extracellular nucleotides was that of Lew and Dearnaley (2000), who
demonstrated that both eADP and eATP could induce large membrane
depolarization changes in root hairs of Arabidopsis thaliana within sec-
onds after the application. Phosphate application had no effect, negating
the explanation that the depolarization was the result of phosphate re-
leased by hydrolysis of the applied nucleotides. Applied ATP, GTP, and
ADPallinducedlargedepolarizations,butAMP,TTP,andCTPdidnot.
Dose-response tests revealed that half-maximal depolarization happened
at0.4mMforATP,butatonly10µMforADP,indicatingthateADPwasthe
more effective inducer of this response. Given that in animals nucleotide
binding induces increased [Ca 2+ ] cyt , whether the receptor is either a P2X
or a P2Y type, the authors tested for a change in [Ca 2+ ] cyt . Using dextran-
conjugated calcium green as the reporter of
[Ca 2+ ] cyt ,theyreportedno
change in [Ca 2+ ] cyt inducedbyeithereATPoreADP.Anincreasein[Ca 2+ ] cyt
would be expected to decrease cytoplasmic streaming in the root hairs, but
applied nucleotides had no effect on this, either. Interestingly, eADP, but
not eATP, induced a slight (22-38%) increase in root hair growth.
Even 10 µM ADP is a much higher concentration than would be expected
in the ECM of any plant or animal cell, so Lew and Dearnaley (2000)
speculated that the most likely naturally occurring situation that would
expose root hairs to high concentrations of eATP would be root wounding,
which would release cytoplasmic ATP to the outside of the cell. Several
authors have measured cytoplasmic ATP concentrations at between 1 and
2 mM (Gout et al. 1992). Although ATP released from root cells by wounding
would be rapidly hydrolyzed by wall-localized apyrases and phosphatases,
itcouldbeexpectedtoreachandremainabove10µMlongenoughto
induce membrane depolarization changes.
If extracellular nucleotides were activating receptors in root hair cells,
their failure to induce changes in the [Ca 2+ ] cyt of these cells suggested that
the signaling pathways they induced were different from those induced by
purinoceptors in animals. Alternatively, it was possible that the experimen-
tal setup used by Lew and Dearnaley was not sensitive enough to detect the
changes in [Ca 2+ ] cyt induced by eATP and eADP. Demidchik et al. (2003)
tested this possibility by using a very different methodology to assess the
effects of extracellular nucleotides on [Ca 2+ ] cyt .Theyusedtransgenic Ara-
bidopsis plants constitutively expressing apoaequorin (Knight et al. 1996).
When these plants take up the luminophore coelenterazine, the apoae-
quorin they are expressing is converted to the bioluminescent calcium
 
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