Agriculture Reference
In-Depth Information
probe, extracellular fluxes have been measured around
P. rhoeas
pollen tubes and
there is good evidence that Ca
2
+
influx occurs in the shanks of normally growing
tubes (20-100 mm behind the pollen tube tip) (Kuhtreiber & Jaffe, 1990; Smith
et al.
,
1994). This is the region in which [Ca
2
+
]
i
transients are seen in the SI response and
raises the possibility that the SI-related transient might be generated through uptake
of extracellular calcium. Experimental evidence that this is indeed the case has been
gained using the
in vitro
bioassay and measuring extracellular Ca
2
+
fluxes 50 mm
behind the pollen tube tip. Challenging pollen tubes with incompatible S-proteins
was found to stimulate influx of Ca
2
+
13.6-fold over a time period comparable to
the increases in [Ca
2
+
]
i
observed by imaging (Franklin-Tong
et al.
, 2002). A small
increase in Ca
2
+
influx was also seen when compatible S-protein was used in the
assay, but was not comparable to that seen in the incompatible challenge. These data
provide convincing evidence that influx of extracellular Ca
2
+
plays a major role in
generating the [Ca
2
+
]
i
transient in the SI response.
10.3.3.2 Protein kinase activity and the SI response
A number of studies have been carried out to determine what lies downstream of the
[Ca
2
+
]
i
transient in this signalling pathway. One obvious avenue to explore is that
of protein phosphorylation and a number of pollen proteins have been identified that
exhibit either an increase or a decrease in phosphorylation when challenged with
self-S-protein (Rudd
et al.
, 1996).
Two proteins that are specifically phosphorylated when pollen tubes are chal-
lenged with self-S-protein have been characterised in some detail. A 26-kDa pollen
protein (with a pI of 6.2) termed p26 is phosphorylated within 90 s of challenging
pollen with self-S-protein, with a further increase occurring at 400 s. This phos-
phorylation is Ca
2
+
-dependent and coincides with the transient increase in [Ca
2
+
]
i
on treatment with self-S-protein, suggesting that the [Ca
2
+
]
i
transient may directly
stimulate phosphorylation of p26 through activation of a Ca
2
+
-dependent protein
kinase (Rudd
et al.
, 1996). Recently, p26 was cloned and has been found to share
80-90% amino acid identity with plant-soluble inorganic pyrophosphatases (Rudd
& Franklin-Tong, 2003). Biochemical assays using recombinant p26 protein indi-
cate that it does indeed possess pyrophosphatase activity and that this activity is
dependent on Mg
2
+
and inhibited by high [Ca
2
+
]
i
,asexpected of an enzyme of this
class (Rudd & Franklin-Tong, 2003). Further, there is a large decrease in pyrophos-
phatase activity in crude pollen tube extracts at nanomolar Ca
2
+
concentrations
that would allow p26 phosphorylation (Rudd & Franklin-Tong, 2003). Hence, the
activity of p26 is almost certainly altered in the SI response. Soluble inorganic
pyrophosphatases play an important role in cellular biosynthesis. They have been
shown to be involved in the generation of both ATP, which drives cellular reactions
and biopolymers required for the synthesis of membranes and cell walls (Coop-
erman
et al.
, 1992). Given these functions it is not difficult to envisage a pivotal
role for p26 in the SI response and it has been proposed that the calcium-induced
phosphorylation of p26 leads to a reduction in pyrophosphatase activity, causing a
depletion of biopolymers (long-chain carbohydrates and proteins) and ultimately a
