Plant Physiol. (1994) 106: 1381-1387
The 58-Kilodalton Calmodulin-Binding Glutamate
Decarboxylase I s a Ubiquitous Protein in Petunia Organs and
I t s Expression I s Developmentally Regulated'
Yali Chen, Gideon Baum, and Hillel Fro"*
Department of Plant Genetics, Weizmann Institute of Science, 761O 0 Rehovot, Israel
et al., 1984); heat shock (Mayer et al., 1990); and water stress
(Rhodes et al., 1986), which is consistent with this role.
Moreover, GAD activity is enhanced at relatively acidic pH
(Snedden et al., 1992; Crawford et al., 1994). It was also
suggested that transamination of a-ketoglutarate by GABA
(producing succinic semialdehyde and glutamate) could regulate carbon flow through the tricarboxylic acid cycle by
bypassing the direct conversion of a-ketoglutarate to succinate, a step that may be inhibited under certain physiological
situations (Dixon and Fowden, 1961). In addition, GABA can
be transaminated even more effectively with pyruvate to
succinic semialdehyde and Ala (Dixon and Fowden, 1961;
Streeter and Thompson, 1972b).
GABA has also been postulated to have a role in nitrogen
metabolism and storage in plants. High molecular mass
GABA conjugates account for more than 6% of the dry weight
of nitrogen-fixing nodules of legumes (Larher et al., 1983).
GABA in nutrient solutions can also function as a sole nitrogen source for plant growth (Bames and Naylor, 1959). In
addition, GABA is an obligatory intermediate in the assimilation of nitrogen from putrescine in a tobacco cell line that
can use putrescine as a sole nitrogen source ( B a h t et al.,
1987).
The mechanisms regulating GAD activity in vivo are still
unknown. GAD activity can be stimulated by lowering cytosolic pH (Crawford et al., 1994). However, reduction of
cytosolic pH is not necessary for stimulation of GABA synthesis (Crawford et al., 1994). This suggests that mechanisms
other than cytosolic pH are also involved in the regulation of
GAD activity. A variety of environmental stresses cause
elevations of GAD activity and cytosolic calcium levels. Thus,
Wallace et al. (1984) suggested that GAD may be regulated
by calcium signaling pathways. Recently, Reggiani et al.
(1993) showed that wheat root GAD is activated in response
to treatments with ABA. ABA can itself induce the elevation
of calcium in plant cells (McAinsh et al., 1990; Gilroy and
Jones, 1992; Bush et al., 1993). We recently cloned a cDNA
encoding a Ca2'-dependent CaM-binding GAD from petunia
(Petunia hybrida) (Baum et al., 1993).This finding is consistent
with the potential role of calcium signaling in the control of
GABA synthesis in plants. This calcium-dependent CaMbinding aspect of plant GAD was previously unknown.
GAD activity and GABA have been detected in virtually
A cDNA coding for a 58-kD calcium-dependent calmodulin
(CaM)-binding glutamate decarboxylase(GAD) previously isolated
in our laboratory from petunia (Petunia hybrida) (G. Baum, Y.
Chen, T. Arazi, H. Takatsuji, H. Fromm [1993] J Biol Chem 268:
19610-19617) was used to conduct molecular studies of GAD
expression. GAD expression was studied during petunia organ
development using the GAD cDNA as a probe to detect the GAD
mRNA and by the anti-recombinant GAD serum to monitor the
levels of GAD. GAD activity was studied in extracts of organs in
the course of development. l h e 58-kD CaM-binding GAD is expressed in all petunia organs tested (flowers and all floral parts,
leaves, stems, roots, and seeds). The highest expression levels were
in petals of open flowers. Developmentalchanges in the abundance
of GAD mRNA and the 58-kD GAD were observed in flowers and
leaves and during germination. Moreover, developmental changes
in GAD activity in plant extracts coincided in most cases with
changes in the abundance of the 58-kD GAD. We conclude that
the 58-kD CaM-binding GAD i s a ubiquitous protein in petunia
organs and that i t s expression is developmentally regulated by
transcriptional and/or posttranscriptional processes. Thus, GAD
gene expression i s likely to play a role in controlling the rates
of GABA synthesis during petunia seed germination and organ
development.
GAD catalyzes the conversion of glutamate to GABA. The
presence of GAD activity and GABA in plants has been
known for at least half a century (refs. in Satyanarayan and
Nair, 1990). However, their roles are still obscure. GABA
functions in animals as a major inhibitory neurotransmitter
by modulating the conductance of ion channels (Zhang and
Jackson, 1993). The suggestions raised to explain the role of
GABA in plants have not addressed the possibility that GABA
may function as a regulator of ion channels as in other
eukaryotes.
Decarboxylation of glutamate to GABA with consumption
of a proton has been suggested as a mechanism to stabilize
cytosolic pH in plant cells during stresses that lead to cytoplasmic acidification (Snedden et al., 1992; Crawford et al.,
1994). The synthesis and accumulation of GABA increase
rapidly in response to anaerobiosis (Streeter and Thompson,
1972a);mechanical shock, cold shock, and darkness (Wallace
This research was supported by a grant to H.F. from the Ministry
of Science and Technology, Israel, and the Gesellschaft fur Biotechnologische Forschung, GBF Braunschweig, Germany.
* Corresponding author; fax 972-8-469124.
Abbreviations: CaM, calmodulin; GABA, y-aminobutyric acid;
GAD, glutamic acid decarboxylase.
1381
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1994 American Society of Plant Biologists. All rights reserved.
1382
Chen et al.
all tissues of numerous plants (Satyanarayan and Nair, 1990).
However, characterization of the GAD enzyme(s)responsible
for this ubiquitous GABA synthesis in plant tissues remains
incomplete. To leam about the possible involvement of the
58-kD CaM-binding GAD in catalyzing GABA synthesis in
different plant organs, we analyzed its expression profile in
different petunia organs during development. We detected
the 58-kD CaM-binding GAD in all petunia organs. Furthermore, we found that transcriptional and/or posttranscriptional processes are involved in regulating GAD expression
and thus may play a role in controlling GABA synthesis in
petunia.
MATERIALS AND METHODS
Plant Material and Growth
Petunia hybrida (var Mitchell) plants were grown in a
greenhouse at 22 to 25OC, 16 h light/8 h dark. Plant material
was collected into liquid nitrogen and stored at -8OOC. For
germination studies, petunia seeds were soaked for 6 h in
water after which they were plated on wet filter paper and
kept under a 16-h light/8-h dark cycle under cool-white
fluorescent light (approximately 50 pE m-' s-').
DNA Isolation and Southern Blot Hybridization
Genomic DNA was extracted from petunia leaves as described (Dellaporta et al., 1983). Samples of DNA (about 60
pg) were digested with the indicated restriction enzymes (100
units each) at 37OC overnight and separated on a 0.8%
(w/v) agarose-Tris acetate-EDTA gel (Sambrook et al., 1989),
stained with ethidium bromide, and blotted onto Genescreen
Plus (NEN) nylon membranes. The blot was prehybridized
in a solution containing 10% (w/v) PEG (mol wt 6000), 50%
(v/v) formamide, 5X SSPE (Sambrook et al., 1989), 2%
(w/v) SDS for 6 h at 42OC, then hybridized under the same
conditions overnight with a random-primed 32P-labeled
probe (Feinberg and Vogelstein, 1983).
Plant Physiol. Vol. .06,1994
Protein Extractions from Plant Tissues
Plant material was frozen in liquid nitrogen and ground
under liquid nitrogen to a fine powder in a mortar. Sodium
ascorbate and polyvinylpolypyrrolidone (100 mg g-' plant
material) were added, and proteins were extracted by further
grinding in extraction buffer (4 mL g-' plant material) containing 100 m Tris-HC1, pH 7.5, 10% (v/v) glycerol, 1 m
Na2-EDTA, 1 r m PMSF, 2.5 pg mL-' leupeptin and antipain
(Sigma). The homogenate was centrifuged at 4OC, 30,OOOg
for 30 min and the supematant was collected. Protein
concentrations were determined with a Bradford reagent
(Bio-Rad).
lmmunodetection of GAD on Western Blots
Protein samples were separated on SDS-PAGE and transferred to nitrocellulose for immunodetection as described
(Baum et al., 1993). The anti-GAD serum raised against a
recombinant GAD was the same as that described by Baum
et al. (1993).
Determination of GAD Activity
Plant extracts were passed through Sephadex 1;-50 columns and reactions were performed with L-[LJ-'~C]G~U
(250
mCi "01-';
Amersham) incubated with protein extracts as
described (Baum et al., 1993). Amino acids were extracted
and fractionated by TLC as described (Baum et d.,1993).
[I4C]GABAlevels were determined by exposing TLC plates
to a BAS-111s Imaging Plate (Fuji Photo Film Co.) and analyzing radioactive [14C]GABA in a Fujix BAS1000 Bio-Imaging
Analyzer. Quantitation of [14C]GABA levels was done by
including samples containing different amounts of a ["C]GABA standard (Amersham) on each TLC plate. After quantitative analysis, TLC plates were exposed to Kodak XAR
films. The position of [14C]GABA on TLC plates was determined according to the mobility of a [l4CJGABAstandard
(Amersham), which was treated in the same buffers and
extraction solutions as the other samples.
RESULTS
RNA Extractions and Analysis on Northern Blots
Genomic Organization of Petunia GAD Genes
.RNA was extracted from plant tissues according to
Logemann et al. (1987) except that RNA pellets were dissolved in 7 M urea, 2% sarkosyl and extracted once with
phenol and then with chloroform:isoamylalcohol(24:1,v/v).
The aqueous phase was collected and RNA was precipitated
and kept at -7OOC. RNA samples were fractionated on
formaldehyde-agarose gels as described (Sambrook et al.,
1989) and transferred to Genescreen Plus membranes according to the manufacturer's guidelines. Membranes were
incubated in a prehybridization solution containing 50%
formamide (v/v), 4X SSC, 1 X Denhardt's solution (Sambrook
et al., 1989), 10% (w/v) PEG (mol wt 6000), 1%(w/v) SDS,
and salmon-sperm DNA (100 pg mL-'; sonicated and boiled)
for 6 h at 42OC. Nick-translated DNA probes were added for
hybridizations for 16 h. Washing of membranes was performed as described (Sambrook et al., 1989).
The petunia GAD cDNA clone was isolated front a cDNA
library of petals (Baum et al., 1993). Prior to analyzing the
expression of the 58-kD GAD, we assessed the number of
genes coding for GAD. Total petunia leaf DNA was digested
with restriction endonucleases, size-fractionated, and transferred to Genescreen Plus membranes (NEN). 32P-labeled
GAD cDNA fragments were used as probes to derect GAD
genomic fragments (Fig. 1, A and B). A single EcoRV fragment
was detected when the whole cDNA was used a:; a probe
(Fig. 1A).Two hybridizing fragments were found after BamHI
digestion, as would be the case for a single-copy ,gene that
contains a single BamHI site in the coding sequence (Baum et
al., 1993). Hind111 and EcoRI digests resulted in a few smaller
hybridizing fragments. These could be interpreted either as
an indication of the presence of more than one GAD-like
gene or as subfragments of a single gene that contains introns
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1994 American Society of Plant Biologists. All rights reserved.
Expression of the 58-kD Glutamate Decarboxylase in Petunia
kb
23.1
—
9.4
—
1383
However, the 58-kD GAD protein continued to accumulate
(per total protein) after levels of its mRNA began declining.
The 58-kD GAD reached maximal levels in open flowers.
GAD activity measured in whole-flower extracts also increased during flower development (Fig. 2, A and B; Table
I). However, the overall changes in GAD activity were smaller
than changes in the abundance of GAD (2.5- versus 10-fold,
respectively), suggesting that factors other than GAD abundance also affect GAD activity.
6.5 _
4.4
—
2.3
—
Figure 1. Organization of CAD-related genes in the petunia genome. Restriction endonuclease digests of total DMA from petunia
leaves were hybridized with the following 32P-labeled DNA fragments as probes: A, the complete CAD cDNA; B, a 450-bp DNA
fragment of CAD cDNA from the 5' terminus to the BamHI site (cf.
Baum et al., 1993). X-Phage DNA digested with H/ndlll was used
as a size marker.
(with no EcoRI and Hi'ndlll endonuclease cleavage sites in
the coding sequence; Baum et al., 1993). To resolve this
ambiguity, another genomic Southern blot was probed with
a 32P-labeled 450-bp DNA fragment that included only the
region from the 5' terminus of the GAD cDNA up to the
unique BamHI site (Baum et al., 1993). A single DNA band
was detected in each of the EcoRV, EcoRI, and BflmHI endonuclease digests. These bands correspond in size to fragments
detected with the full cDNA probe in Figure 1A.
We also cloned genomic fragments containing the GAD
coding sequence. Their preliminary analysis confirms the
existence of introns (not shown). Therefore, the petunia 58kD CaM-binding GAD is most likely encoded by a single
gene containing introns. However, the existence of more
distantly related petunia GAD genes that did not hybridize
to the whole cDNA probe or to the smaller cDNA probe
cannot be excluded.
Flower size (cm)
in
o
5 3
6
I
oi
GAD mRNA
Northern blot
GAD protein
Western blot
GABA
0.5
TLC
1.0
2.0
2.0-7.0
7.0
(open)
Flower size (cm)
Figure 2. Expression of the 58-kD CAD in developing petunia
flowers. Whole flowers at different stages of development ranging
in length from 0.5 to 7.0 cm (fully expanded open flowers) as
indicated were collected and RNA and proteins were extracted.
Flowers ranging in length from 2.0 to 7.0 cm (but before opening)
were pooled and tested as a single sample (indicated as 2.0-7.0).
Protein samples (20 jtg each) were fractionated by SDS-PACE and
transferred to nitrocellulose membranes for immunoblot analysis.
Total RNA samples (10 Mg each) were loaded for northern blot
Developmental Regulation of GAD Expression in Flowers
analysis. A, Autoradiogram of a northern blot showing the CAD
mRNA signal, a western blot showing the 58-kD CAD, and a TLC
GAD mRNA was hardly detected in the youngest flowers
plate showing the ['4C]CABA produced within 1 h of in vitro
tested (0.5 cm long), whereas striking changes in the abundecarboxylation of L-[U-'4C]Glu (see "Materials and Methods"). B,
dance of GAD mRNA were detected during flower developRelative abundance of the CAD mRNA, the 58-kD GAD, and CAD
ment (Fig. 2, A and B) and the highest levels of GAD mRNA
activity during flower development. The highest level of each
were found in 2.0-cm-long flowers, about 10 times more than
parameter in this experiment was set at 100%. Quantitation of
the levels in 0.5-cm flowers. The levels of GAD mRNA then
mRNA and protein levels was determined by scanning films with a
declined rapidly; in fully expanded open flowers the levels
Molecular Dynamics (Sunnyvale, CA) 300A Computing Densitomwere similar to those in 0.5-cm-long flowers. Increases in the
eter. Quantitation of CAD activity was performed as described in
abundance of the 58-kD GAD protein appeared in parallel
"Materials and Methods." Relative levels of CAD mRNA and protein
were the averages of two experiments. Bars indicate SE values.
with the increase in the abundance
of
the
GAD
mRNA.
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1994 American Society of Plant Biologists. All rights reserved.
Chen et al.
1384
Plant Physiol. Vol. 106,1994
Table 1. Changes in CAD activity in petunia extracts during organ
development
CAD Activity'
Organ
From
To
pmol GABA p
protein h~
Leaf
Whole flowerb
Sepal
Petal-tube
Petal-limb
Stamen
Ovary
Style
Seed germination
140
467
541
243
423
30
202
60
16
Pe •—Tu
601
1016
676
601
1142
36
322
238
39
Se
B
a
CAD activity was determined as described in "Materials and
Methods." The values indicated represent minimal and maximal
activities measured in the course of organ development.
b
Whole flowers ranged in length from 0.5 to 7.0 cm as in Figure 2.
Floral parts were dissected from two stages of flower development:
2.0 cm and 7.0 cm long (open flowers). See Figure 3 for a schematic
c
presentation of petunia floral organs.
Values represent the
activity in extracts from whole dry seeds and after 120 h of germination. However, CAD activity in whole seed extracts declined
during the first 72 h of germination and increased after that period
(cf. Fig. 6).
GAD protein
-—
Western blot
GABA
TLC
Limb Development
We dissected fully expanded, open petunia flowers and
analyzed the expression of GAD in the different floral organs
(Fig. 3). Petals were dissected into rubes (the lower cylindrical
part) and limbs (the upper expanding part) (see Fig. 3A, a
schematic presentation of petunia floral parts; modified from
Van der Krol and Chua, 1993), because differences in the
expression of genes between the two parts have been reported
(Takatsuji et al., 1992). The highest abundance of the 58-kD
GAD and the highest GAD activity were apparent in the
limbs (Fig. 3B). The abundance of the 58-kD GAD and GAD
activity (per total protein) were lower in extracts of sepals,
tubes, ovaries, and styles (see Table I). Stamens contained
low levels of the 58-kD GAD (not shown) and GAD activity
(Table I). Petals make up about 70% of the biomass of open
flowers of P. hybrida var Mitchell (our analysis). Thus, the
stimulation of GAD expression observed in whole flowers
during development (cf. Fig. 2) is probably a manifestation
of GAD expression in petals. Indeed, we observed a developmental stimulation of GAD expression in limbs (Fig. 3C).
GAD Expression during Leaf Development
Flower Length (cm)
c
I
CO
GAD protein
GABA
Western blot
tit
TLC
Figure 3. Expression of the 58-kD CAD in floral organs. A, Schematic presentation of a longitudinal section of a mature petunia
flower (modified from Van der Krol and Chua, 1993). Floral parts
are as follows: Se, sepal; Pe, petal; Li, limb; Tu, tube; St, stamen;
Sy, style; Sg, stigma, Ov, ovary. B, Organs from fully expanded
open flowers were dissected and proteins were extracted. Analysis
of protein on western blots and off CABA synthesis was as described
in the legend to Figure 2. A western blot analysis of the 58-kD CAD
protein and an autoradiogram of a TLC plate showing the [14C]CABA produced within 1 h of in vitro decarboxylation of [14C]glutamate are presented. C, Developmental regulation of CAD
expression in limbs of flowers at different stages of development
(lengths of flowers from which limbs were dissected are indicated
in cm). CAD protein levels and CABA synthesis were analyzed as
described in the legend to Figure 2.
GAD expression was analyzed in leaves at different stages
of development (Fig. 4, A and B). The levels of the GAD
mRNA and of the 58-kD GAD increased in parallel during
leaf development. Maximal GAD mRNA levels were detected
in 6.0-cm-long leaves, and these were about 2.5-fold more
than in the youngest leaves (0.5-1.0 cm long). A slight decline
in the levels of the GAD mRNA and protein was observed
in 9.0-cm-long leaves. Changes in GAD activity in leaf
extracts coincided with changes in GAD level (Fig. 4B;
Table I), both showing about 5-fold increases during leaf
development.
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1994 American Society of Plant Biologists. All rights reserved.
1385
Expression of the 58-kD Glutamate Decarboxylase in Petunia
Leaf development
3
GAD mRNA
GAD protein
Northern blot
— <
Western blot
tiff
GABA
L2
L3
L4
LS
TLC
L6
Leaf development
Figure 4. Expression of the 58-kD CAD in developing leaves.
Protein and mRNA from leaves at different stages of development
were extracted and analyzed as described in the legend to Figure
2. The lengths of leaves (cm) were: L1, 0.5 to 1.0; L2, 1.0 to 2.0; L3,
2.0 to 3.0; L4, 3.0 to 5.5; L5, 6.0; L6, 9.0. A, Autoradiogram of a
northern blot showing the CAD mRNA signal, a western blot
showing the 58-kD CAD protein band, and a TLC plate showing
the [14C]CABA produced in a 1-h reaction with radiolabeled glutamate. B, Relative changes in the CAD mRNA, 58-kD CAD, and
CAD activity, determined as described in the legend to Figure 2.
The highest level of each parameter in this experiment was set at
100%. Each point on the activity plot is the average of two experiments. Bars indicate SE values.
increased and GABA accumulated (Inatomi and Slaughter,
1971; Vandewalle and Olsson, 1983). It was proposed that
the GABA-shunt pathway provides carbon skeletons for oxidation in the tricarboxylic acid cycle, a step that is of particular importance during germination (Vandewalle and Olsson,
1983).
To examine the possibility that the 58-kD GAD may be
involved in metabolic processes in seeds, we analyzed its
expression in dry seeds and during the first 5 d of germination. GAD mRNA was undetectable in dry seeds, but the 58kD GAD was clearly apparent. This may be explained by the
deposition of GAD in seeds during fruit development. We
indeed detected the 58-kD CaM-binding GAD in developing
petunia fruits (not shown).
GAD activity in dry seed extracts was about 10% of that
in young-leaf extracts used as a control (Fig. 6; Table I). After
12 h of germination, a slight decline in the abundance of the
58-kD GAD protein and in GAD activity was observed. At
48 h of germination, the abundance of the 58-kD GAD was
minimal. Two other protein bands (48 and 66 kD) crossreacted with the anti-GAD serum in dry seeds and during
the first 60 h of germination. The levels of these two proteins
remained maximal as long as the abundance of the 58-kD
protein was minimal. At 60 h of germination, a decline in the
abundance of the 48- and 66-kD proteins was apparent,
whereas the abundance of the 58-kD GAD increased slightly.
At this stage, the major seed proteins (Fig. 6, open arrowheads) had significantly declined and the root had already
emerged from the seed coat (not shown). At 72 h of germination, the 48- and 66-kD protein bands were hardly detected, whereas the GAD mRNA and the 58-kD protein were
more abundant. At this stage the major seed proteins were
no longer apparent. At 120 h of germination significant
increases in the abundance of the GAD mRNA, the 58-kD
GAD, and GAD activity were apparent, the cotyledons had
just emerged, and the small and large subunits of Rubisco
(Fig. 6, SS and LS, respectively) were detected at that time.
DISCUSSION
In this work we studied the expression of the 58-kD CaMbinding GAD in different petunia organs and assessed the
GAD Expression in Roots and Stems
The expression of GAD was tested in roots and stems of
petunia plants together with samples from leaves (4.5-5.5
cm long) and young flowers (2.0 cm long) from the same
plants (Fig. 5). The results show that the 58-kD GAD is
expressed in all organs tested. The protein level and GAD
activity in stem extracts was higher than in leaves, roots, and
young flowers (but lower than in open flowers; comparison
not shown).
GAD protein
GABA
>—
•t t
-
Western blot
TLC
Figure 5. Expression of the 58-kD CAD in various organs of petunia
plants. Roots, stems, leaves (4.5-5.5 cm long), and young flowers
(2.0 cm long) were dissected and analyzed as described in the
Special attention has been given by several investigators
legend to Figure 2. A representative western blot showing the 58to the possible roles of GAD and GABA in seeds during
kD CAD protein band and a TLC plate showing the [14C]CABA
germination. GAD activity was detected at very early stages
produced within 1 h of in vitro decarboxylation of [14C]glutamate
of germination, and at the onset Downloaded
of growth GAD
activity
presented.
from on June 18, 2017are
- Published
by www.plantphysiol.org
Copyright © 1994 American Society of Plant Biologists. All rights reserved.
GAD Expression during Germination
1386
Chen et al.
Plant Physiol. Vol. 106, 1994
mRNA levels and protein levels in leaves, as opposed to
petals, suggests that posttranslational regulation of GAD may
differ in petals and leaves and may play a role in controlling
GAD levels.
-LS
Increased levels of the 58-kD GAD during organ development and seed germination coincided with increases in the
in vitro-measured GAD activity. These results suggest that
31.0 the 58-kD CaM-binding GAD is responsible for a major
Protein stain
portion of the overall GAD activity in petunia extracts. However, we cannot exclude the possibility that other GAD
21.5 —
proteins that are not detected by our antibodies have a similar
expression profile and also contribute to the overall GAD
activity. This could be one explanation for the fact that during
flower development the level of the 58-kD GAD in young
66kDaflowers was about 10% of that in open flowers, although the
58 KOa GAD Western blot
activity in young flower extracts was about 40% of that in
48kOaopen flowers. A summary of the overall GAD activity in
plant extracts during organ development and germination is
GABA
'J
TLC
presented in Table I.
We note that the activity in plant extracts does not necessarily reflect relative activities in vivo, which may be reguGAD mRNA
Northern blot
lated by several effectors such as cytosolic pH (Snedden et
al., 1992; Crawford et al., 1994), ABA (Reggiani et al., 1993),
and calcium signaling via CaM (Baum et al., 1993). However,
Figure 6. Expression of the 58-kD CAD during germination. Seeds
recent findings in our laboratory show that transgenic plants
were collected prior to soaking (0 h) and at the times indicated after
overproducing the 58-kD CaM-binding GAD under the tranthe beginning of the soaking period. Analysis of the CAD mRNA,
scriptional control of the 35S cauliflower mosaic virus prothe 58-kD CAD, and CAD activity were performed as described in
moter possess higher rates of GABA synthesis in vivo. Thus,
the legend to Figure 2 (0.5- to 1.0-cm leaves from flowering petunia
the overall GAD activity in plants can be regulated in part
plants were used as a control). Samples containing 20 jig of total
by
GAD abundance (Baum et al., 1994).
soluble protein were separated by SDS-PAGE and stained with
GAD in seeds may have unique features when compared
Coomassie brilliant blue. An identical gel was used for electrotransto other organs. In addition to the 58-kD GAD, two other
fer of proteins to a nitrocellulose membrane and detection of the
58-kD CAD with anti-recombinant GAD serum. The 58-kD CAD
seed proteins cross-reacted with the anti-GAD serum (48 and
band and two other proteins detected with the anti-CAD serum
66 kD; cf. Fig. 6). The identity of these proteins is still obscure,
are indicated with their respective molecular masses (58, 48, and
but it is interesting that they showed an expression profile
66 kD; full arrows on the left). Arrows on the right show the Rubisco
opposite from that of the 58-kD GAD during germination
small and large subunits (SS and LS, respectively). Major seed
and that they were detected only in developing fruits (not
proteins that disappeared during germination are indicated by open
shown), in dry seeds, and during the first 60 h of germination.
arrowheads. An autoradiogram of a TLC plate showing the [14C]Inatomi and Slaughter (1975) described the occurrence of
CABA produced in 1 h of in vitro decarboxylation and a northern
different forms of high molecular mass GAD complexes in
blot probed with the 32P-labeled CAD cDNA are presented below
embryos and in roots of barley. However, in our study GAD
the western blot.
activity during petunia seed germination coincided with the
abundance of the 58-kD protein (Fig. 6).
With respect to the possible function of CaM in seeds
relationships between changes in the levels of the correduring germination, it is interesting that recently a number
sponding mRNA, protein, and activity during organ
of small proteins purified from radish seeds were identified
development.
as potent CaM antagonists (Polya et al., 1993). Moreover, a
In the course of development, increases in the abundance
significant decrease in these antagonists and a simultaneous
of the 58-kD GAD coincided with increases in the levels of
increase in CaM levels were observed during early phases of
the GAD mRNA (cf. Figs. 2, 4, and 6). These results suggest
germination, implying that CaM-regulated processes are acthat during petunia organ development transcriptional and/
tivated (Cocucci and Negrini, 1988). It is conceivable that the
or posttranscriptional regulatory processes are involved in
58-kD CaM-binding GAD in petunia is one of the enzymes
controlling GAD expression. In petals, the 58-kD GAD conthat are activated by Ca2+/CaM in the course of germination.
The striking increase in the expression of the 58-kD GAD
tinued to accumulate after its mRNA began declining, so that
during flower development and its accumulation to high
in open flowers the GAD mRNA was hardly detected but
levels in petals deserve further attention. We have no obvious
GAD abundance was maximal. This observation indicates
explanation for these results. A speculative link between
that in petals, the 58-kD GAD is a relatively stable protein.
GAD activity and petal development may be found in the
Conversely, in large leaves, a decline in GAD mRNA levels
fact that the pH of petal cells is an important factor in
coincided with a decline in the abundance of the 58-kD GAD
regulating
pigmentation (Sink, 1984). Furthermore,
and in GAD activity (Fig. 4). ThisDownloaded
apparent coupling
between
from on June 18, 2017 - Published byflower
www.plantphysiol.org
Germination (hr)
'O
12 24 48 60 72 120\.«at
Copyright © 1994 American Society of Plant Biologists. All rights reserved.
Expression of the 58-kD Glutamate Decarboxylase in Petunia
genes responsible for controlling petal pH i n petunia have
been isolated recently (Chuck et al., 1993), but their identity
has not been revealed. Finally, because GAD h a s been reported to be stress activated (Wallace et al., 1984; Rhodes et
al., al., 1986; Mayer e t a]., 1990), it is noteworthy that other
stress-induced proteins are expressed i n flowers. These include pathogenesis-related proteins (Lotan e t al., 1989) and
heat-shock proteins such as HSP70 (Y. C h e n and H. Fromm,
unpublished observations).
In summary, we found that t h e 58-kD CaM-binding GAD
is a ubiquitous protein in petunia organs and that its expression is developmentally regulated by transcriptional and/or
posttranscriptional processes. These regulatory mechanisms
are likely to b e involved in controlling GABA synthesis during
petunia development. It is not yet known how the regulation
of GAD gene expression plays a role in concert with other
potential effectors of GAD activity.
ACKNOWLEDGMENTS
We thank Ms. Dvora Dolev for excellent technical assistance and
Drs. Esra Galun, Jonathan Gressel, Robert Fluhr, Gad Galili, Adi
Avni, Julio Salinas, and Hiroshi Takatsuji for critical reading of the
manuscript.
Received June 14, 1994; accepted August 6, 1994.
Copyright Clearance Center: 0032-0889/94/106/1381/07.
LITERATURE CITED
B a h t R, Copper G, Staebell M, Filner P (1987) N-Caffeoyl-4amino-n-butyric acid, a new flower-specific metabolite in cultured
tobacco cells and tobacco plants. J Biol Chem 262 11026-11031
Barnes RL, Naylor AW (1959) Effect of various nitrogen sources on
growth of isolated roots of Pinus serotina. Physiol Plant 12: 82-89
Baum G, Chen Y, Arazi T, Takatsuji H, Fro” H (1993) A plant
glutamate decarboxylasecontaining a calmodulin-binding domain:
cloning, sequence and functional analysis. J Biol Chem 268
19610-19617
Baum G, Dolev D, Fromm H (1994) High GABA levels in transgenic
plants overexpressing the petunia 58 kDa CaM-binding glutamate
decarboxylase (abstract No. 170). Plant PhysiollO5 5-41
Bush DS, Biswas AK, JonesRL (1993) Hormonal regulation of Ca2+transport in the endomembrane system of the barley aleurone.
Planta 189 507-515
Chuck G, Robbins T, Nijjar C, Ralston E, Courtney-GuttersonN,
Dooner HK (1993) Tagging and cloning of a petunia flower color
gene with the maize transposable element Activator. Plant Cell 5
371-378
Cocucci M, Negrini N (1988) Changes in the levels of calmodulin
and of a calmodulin inhibitor in the early phases of radish (Raphanus sativus L.) seed germination. Plant Physiol88: 910-914
Crawford LA, Bown AW, Breitkreuz KE, Guine1 FC (1994) The
synthesis of y-aminobutync acid in response to treatments reducing cytosolic pH. Plant Physiol104 865-871
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version 11. Plant Mol Biol Rep 1: 19-21
Dixon ROD, Fowden L (1961) y-Aminobutyric acid metabolism in
plants. 2. Metabolism in higher plants. Ann Bot 2 5 513-530
1387
Feinberg AP, Vogelstein B (1983) A technique for radiolabeling
DNA restriction endonuclease fragments to high specific activity.
Anal Biochem 132 6-13
Gilroy S, Jones RL (1992) Gibberellic acid and abscisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley
aleurone protoplasts. Proc Natl Acad Sci USA 8 9 3591-3595
Inatomi K, Slaughter JC (1971) The role of glutamate decarboxylase
and y-aminobutyric acid in germinating barley. J Exp Bot 22:
561-571
Inatomi K, Slaughter JC (1975) Glutamate decarboxylase from
barley embryos and roots. Biochem J 147: 479-484
Larher F, Goas G, Le Ruddier D, Gerard J, Hamelin J (1983)
Bound 4-aminobutync acid in root nodules of Medicago sativa and
other nitrogen fixing plants. Plant Sci Lett 2 9 315-326
Logemann J, Schell J, Willmitzer L (1987) Improved method for
the isolation of RNA from plant tissues. Anal Biochem 163 16-20
Lotan T, Ori N, Fluhr R (1989) Pathogenesis-related proteins are
developmentally regulated in tobacco flowers. Plant Cell 1:
881-887
Mayer RR, Cherry JH, Rhodes D (1990) Effects of heat shock on
amino acid metabolism of cowpea cells. Plant Physiol94 796-810
McAinsh MR, Brownlee C, Hetherington AM (1990) Abscisic acidinduced elevation of guard cell cytosolic Ca2+precedes stomatal
closure. Nature 343 186-188
Polya GM, Changra S, Condron R (1993) Purification and sequencing of radish seed calmodulin antagonists phosphorylated by calcium-dependent protein kinase. Plant Physiol 101: 545-55 l
Reggiani R, Aurisano N, Mattana M, Bertani A (1993)ABA induces
4-aminobutyrate accumulationin wheat seedlings. Phytochemistry
3 4 605-609
Rhodes D, Handa S, Bressan RA (1986) Metabolic changes associated with adaptation of plant cells to water stress. Plant Physiol
8 2 890-903
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A
Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY
Satynarayan V, Nair PM (1990) Metabolism, enzymology and possible roles of 4-aminobutyrate in higher plants. Phytochemistry
2 9 367-375
Sink KC (1984) Petunia. In KC Sink, ed, Monographs on Theoretical
and Applied Genetics, Vol 9. Springer-Verlag, New York, pp
49-67
Snedden WA, Chung I, Pauls RH, Bown AW (1992) Proton/
L-glutamate symport and the regulation of intracellular pH in
isolated mesophyll cells. Plant Physiol99 665-671
Streeter JG, Thompson JF (1972a) Anaerobic accumulation of
y-aminobutyric acid and alanine in radish leaves (Raphanus sativus). Plant Physiol49 572-578
Streeter JG, Thompson JF (1972b) In vivo and in vitro studies on
y-aminobutyric acid metabolism with the radish plant (Raphanus
sativus L.). Plant Physiol49 579-584
Takatsuji H, Mori M, Benfey PN, Ren L, Chua N-H (1992) Characterization of a zinc finger DNA-binding protein expressed specifically in Petunia petals and seedlings. EMBO J ll: 241-249
Van der Krol AR, Chua N-H (1993) Flower development in petunia.
Plant Cell 5 1195-1203
Vandewalle I, Olsson R (1983) The y-aminobutync acid shunt in
germinating Sinapis alba seeds. Plant Sci Lett 31: 269-273
Wallace W, Secor J, Schrader LE (1984) Rapid accumulation of
y-aminobutync acid and alanine in soybean leaves in response to
an abrupt transfer to lower temperature, darkness, or mechanical
manipulation. Plant Physiol75 170-175
Zhang SJ, Jackson MB (1993) GABA-activated chloride channels in
secretory nerve endings. Science 259 531-534
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1994 American Society of Plant Biologists. All rights reserved.