in wave features of these oscillations as described
previously for tobacco pollen tubes. Although no
significant difference was recorded in average current intensity between wild-type and glr1.2-1 pollen
tubes, we detected a qualitative difference on the
oscillatory behavior. Oscillating Ca2+ flux represented 59.7% T 4.1 (n = 13) of total Ca2+ fluxes in
wild type, but they represented only 33.2% T 2.3
(n = 13) in pollen tubes from the glr1.2-1 line
(Fig. 3, D and F). This phenotype shows that, as
in tobacco, GLRs are involved in the generation of
Ca2+ influx oscillations in Arabidopsis pollen tubes.
CNQX phenocopied this result, showing no significant difference in the average flux intensity but
significant differences in the oscillation amplitude
(Fig. 3, E and F). These results further confirm that
GLR activity is involved in Ca2+ signaling in the
pollen tube by controlling [Ca2+]cyt through Ca2+
influxes. In particular, the effect of GLRs seems
specific to the modulation of the oscillatory “Ca2+
signature,” a hallmark of Ca2+ signaling in eukaryotic cells (27).
D-Ser plays an active role in pollen tube growth
in vitro. Interestingly, the most active agonist of
GLR activity in pollen tubes of tobacco and
Arabidopsis was D-Ser. GLR1.2 disruption had a similar
effect as decreasing D-Ser–dependent GLR activity
with CNQX. This result was unexpected because
D-Ser is a very rare amino acid, although it is the
subject of active research because of its role as a
neuromodulator in specific neuronal circuits (28).
The activity of a racemase is required to generate
D-Ser from the physiologically inactive L-Ser. We
thus investigated D-Ser formation and effects in
Arabidopsis. As in tobacco, it increased [Ca2+]cyt
measured with the YC3.6 probe (Fig. 4) and
increased Ca2+ influx and oscillation amplitudes
at a physiological concentration (100 mM) (Fig.
5A). The induction of oscillations by D-Ser is
particularly clear on [Ca2+]cyt (Fig. 4D).
So far, only one gene has been identified in the
Arabidopsis genome (SR1 At4g11640) that corresponds to a serine-racemase activity in vitro (29).
According to microarray data, this serine-racemase
is expressed in the pistil, and particularly in the
ovule (30). We cloned the serine-racemase gene
promoter and performed a b-glucuronidase fusion
assay on flowers harvested from transgenic plants
expressing the construct. In three independent lines,
b-glucuronidase was detected all over the pistil but,
as confirmed by the microarray data, was particularly strong in the ovule, especially in the region
close to the micropyle (inset, Fig. 5B). Immunolocalization of D-Ser in the pistil (Fig. 5C; D-Ser in
blue, red is autofluorescence) confirmed the serine-racemase expression pattern. To demonstrate a correlation between the distribution of D-Ser and the
expression of SR1, we characterized an insertion
line for the serine-racemase gene (Fig. 5, D and E).
A very weak D-Ser signal was detected in sr1-1
pistils by immunolocalization (Fig. 5D), demonstrating that serine-racemase synthesizes D-Ser in
plants. We further investigated whether the absence
of normal levels of D-Ser could effect abnormal
pollen tube growth in vivo. Pistils from the sr1-1
W T pollen
G eno mic D N A
J ( pmol.cm-2s-1)
J ( pmol.cm-2.s-1)
J ( pmol.cm-2.s-1)
10 11 12 13 14
lortn o C
Oscillating fluxes (%)
Fig. 3. GLR1.2 is involved in Arabidopsis pollen tube morphogenesis and in Ca2+ apical influx
oscillations. (A) RT-PCR analysis for GLR1.2, Vanguard1 (At4g12730 pollen-specific) (20), AtEXP1
(At2g47040 absent in pollen) (20), and TUB4 expression in wild-type pollen (lane 1) and seedlings (lane 2).
Lane 3 is PCR performed on genomic DNA (positive control). (B) Atglr1.2-1 insertion line showing T-DNA
located within the second exon. The transcript from GLR1.2 was not detected by RT-PCR in inflorescence of
Atglr1.2-1 (inset). Number of seeds per silique in wild-type, Atglr1.2-1 and GLR1.2-antisense plants
(distribution curves, n > 100). (C) Arabidopsis wild-type pollen tube grown in the presence of CNQX (172 mM,
upper panel) and Atglr1.2-1 pollen tube grown in control condition (lower panel). (D) Typical Ca2+-specific
vibrating probe recordings in growing pollen tube of wild-type and Atglr1.2-1. (E) Effect of CNQX (172 mM)
on Ca2+ apical influx in wild-type pollen tubes. (F) Ratio of oscillating Ca2+ flux in Atglr1.2-1 compared with
wild type in the presence of CNQX compared with control condition (wild-type plants).
Fig. 4. D-serine increases
[Ca2+]cyt in Arabidopsis
pollen tubes. (A) Typical
YC3.6 cameleon imaging
in a growing Arabidopsis
pollen tube (n = 10).
Upper and lower panels,
respectively, depict the
minimum (*) and maximum (**) [Ca2+]cyt at the
tip of the same cell. (B)
After D-Ser (5 mM) application, tubes exhibit an
increase in [Ca2+]cyt (n =
7) and an extension of
the gradient toward the
subapical zone. (C and D)
Kymographs from the tubes
presented in (A) and (B), respectively. Each horizontal line of the kymograph illustrates the [Ca2+]cyt values
along a line traced in the middle of the tube at one time point. The slope of the kymograph represents the
growth rate of the tube. Note the increase in [Ca2+]cyt and activation of oscillations by addition of D-Ser.
Color scale represents the fluorescence resonance energy transfer ratio (3 ≈ 100 nM and 6 ≈ 0.5 mM) (3).
line were thus pollinated with wild-type pollen
grains, and pollen tubes were imaged by aniline
blue staining (Fig. 5E). We systematically observed
deformations and branching of wild-type tubes
grown in pistils of the sr1-1 genetic background.
Remarkably, these deformations mimicked both
the phenotype of Atglr1.2-1 or CNQX-treated pol-
len tube grown in vitro and Atglr1.2-1 pollen tube