(Received for publication, March 13, 1995; and in revised form, June 26, 1995)
From the
Calmodulin plays pivotal roles in the transduction of various
Ca-mediated signals and is one of the most highly
conserved proteins in eukaryotic cells. In plants, multiple calmodulin
isoforms with minor amino acid sequence differences were identified but
their functional significances are unknown. To investigate the
biological function of calmodulins in the regulation of
calmodulin-dependent enzymes, we cloned cDNAs encoding calmodulins in
soybean. Among the five cDNAs isolated from soybean, designated as SCaM-1 to -5, SCaM-4 and -5 encoded
very divergent calmodulin isoforms which have 32 amino acid
substitutions from the highly conserved calmodulin, SCaM-1 encoded by SCaM-1 and SCaM-3. SCaM-4 protein produced in Escherichia coli showed typical characteristics of calmodulin
such as Ca
-dependent electrophoretic mobility shift
and the ability to activate phosphodiesterase. However, the extent of
mobility shift and antigenicity of SCaM-4 were different from those of
SCaM-1. Moreover, SCaM-4 did not activate NAD kinase at all in contrast
to SCaM-1. Also there were differences in the expression pattern of SCaM-1 and SCaM-4. Expression levels of SCaM-4 were approximately 5-fold lower than those of SCaM-1 in
apical and elongating regions of hypocotyls. In addition, SCaM-4 transcripts were barely detectable in root whereas SCaM-1 transcripts were as abundant as in apical and elongating regions
of hypocotyls. In conclusion, the different biochemical properties
together with differential expression of SCaM-4 suggest that this novel
calmodulin may have different functions in plant cells.
Calmodulin, a highly conserved and ubiquitous protein in
eukaryotes, mediates Ca signals to various target
proteins(1) . A variety of regulatory enzymes and proteins such
as protein kinases, ion channels, Ca
pumps, nitric
oxide synthetase, inositol trisphosphate kinase, cyclic nucleotide
phosphodiesterase, and NAD kinase are known to be regulated by
Ca
and
calmodulin(2, 3, 4) . While a great deal of
information has been known for biological roles of calmodulin in animal
cells, very little is known about the roles of calmodulin in plant
cells. This is mainly due to the absence of purified
calmodulindependent enzymes and/or their genes in plants. As an effort
to investigate the biological role(s) of calmodulin in plants,
calmodulin genes in various plant species have been cloned and
characterized recently(4) . Interestingly, in Arabidopsis, cDNAs encoding multiple calmodulin isoforms have
been isolated although the degree of sequence divergence is minor, only
6 amino acid differences between the two most divergent isoforms among Arabidopsis calmodulins(5, 6, 7) .
This is very notable because, in animal cells, only a single form of
calmodulin is produced by a calmodulin multigene
family(8, 9) . However, it has not been determined
whether plant calmodulin isoforms have the same biochemical properties
such as calcium-binding abilities and activation of
calmodulin-dependent enzymes. Also the biological role of multiple
calmodulin isoforms in vivo is completely unknown.
To
understand Ca/calmodulin-mediated signal transduction
mechanisms in plants, first we cloned calmodulin cDNAs from soybean.
From these cDNA clones, we have identified two novel divergent
calmodulin isoforms. One of the calmodulin isoforms showed several
distinct characteristics to other highly conserved plant calmodulin
isoforms. Here we describe the structural and functional differences
between the novel divergent calmodulin isoform SCaM-4 and the highly
conserved calmodulin SCaM-1.
where v is the observed rate, V is the maximal activity, [CaM] is the concentration of
added calmodulin, K
is the concentration of
calmodulin required for half-maximal activity, and n is the
Hill coefficient.
Figure 1:
Genomic Southern blot
analysis of soybean calmodulin multigene family. Genomic DNA was
isolated from leaves of mature soybeans. Ten µg of purified genomic
DNA was digested either with EcoRI (E), BamHI (B), or HindIII (H), size
fractionated on a 0.8% agarose gel, and transferred onto a nylon
membrane. The filter was hybridized with a P-labeled
calmodulin coding region probe as described under ``Materials and
Methods.'' M indicates size markers expressed in
kilobases.
Figure 2: Comparison of the deduced amino acid sequences of five soybean calmodulin cDNAs. Deduced amino acid sequences of five SCaMs (this work), barley(33) , and bovine (43) calmodulins were aligned for comparison. Gaps were introduced to maximize homology. Calcium binding ligands were denoted by asterisks (*)(44) , and dashes indicate identical amino acids to those of SCaM-1.
Figure 3: Relationship of soybean calmodulin isoforms to other calmodulins. Thirty-three amino acid sequences of calmodulins from 23 organisms were obtained by GenBank (Release 77) searches. The sequences were aligned and compared to construct a phylogenetic tree by using the neighbor-joining method (see ``Materials and Methods''). ACaM-x indicates six calmodulin cDNAs isolated from Arabidopsis(5, 6, 7) . The bar indicates 0.1 substitutions/site.
Figure 4:
Ca-dependent
electrophoretic mobility shifts of SCaM-1 and -4 proteins. SCaM-1 and
-4 proteins were produced in E. coli using a T7 expression
vector system (15) and purified to homogeniety using
phenyl-Sepharose column chromatography as described under
``Materials and Methods.'' Two µg of purified SCaM
proteins and bovine brain calmodulin (Sigma) were electrophoresed on a
13.5% SDS-polyacrylamide gel either in the presence of 5 mM
CaCl
or 5 mM EGTA in sample buffers. Protein bands
were visualized by Coomassie Brilliant Blue staining. M indicates size markers shown in kilodaltons
(Bio-Rad).
Also, we investigated antigenic differences among the two isoforms and bovine calmodulin. Unpurified antisera raised against SCaM-1 and SCaM-4 in goats were examined for cross-reactivity by immunoblot analysis. As shown in Fig. 5, anti-SCaM-4 antiserum recognized SCaM-4 as expected but not SCaM-1 and bovine calmodulin. In contrast, anti-SCaM-1 antisera recognized SCaM-1 and bovine calmodulin but not SCaM-4, suggesting that SCaM-1 has a more closely related structure to bovine calmodulin than SCaM-4. However, after overexposure of immunoblots, faint cross-reacting bands were observed in both blots, indicating that the two isoforms may have common but very weak antigenic epitopes (data not shown). Thus the two calmodulin isoforms have different major antigenic determinants despite 70% of amino acid identity. Control experiments with anti-bovine brain calmodulin antibody showed a poor cross-reaction with SCaM-4 in contrast to a strong cross-reaction with SCaM-1 as effective as bovine brain calmodulin (data not shown). These data strongly suggest that the multiple amino acid substitutions may confer significant differences on the protein structure of SCaM-4.
Figure 5: Immunoblot analysis of purified SCaM-1 and -4 proteins with antisera raised against SCaM-1 or SCaM-4. An equal amount of protein (50 ng/lane) was electrophoresed on a 13.5% SDS-polyacrylamide gel in the presence of 5 mM EGTA. Fractionated proteins were blotted onto a PVDF (Millipore) membrane and incubated with either unpurified anti-SCaM-1 or anti-SCaM-4 antisera. The immune complexes were visualized by using the ECL system (Amersham) after incubating with horseradish peroxidase-conjugated anti-goat IgG.
Figure 6:
Activation of
Ca/calmodulin-dependent enzymes by SCaM isoforms.
Dose-response curves of PDE and NAD kinase are shown. Data points
represent means of three (for PDE) or five (for NAD kinase) independent
assay results, and error bars represent standard deviations.
Fitted curves are drawn from the Hill equation as described under
``Materials and Methods.'' Panel A, activation of
PDE by SCaM-1 and SCaM-4. PDE assay was done using calmodulin-deficient
bovine heart PDE and cAMP as substrate. Activity of PDE was monitored
with varying amounts of calmodulins and expressed as a relative
activity to that of PDE in the presence of 100 nM activator
SCaM-1. K
values of SCaM-1 and SCaM-4 for PDE
were 7.63 and 6.17 nM, and the Hill coefficients were 1.87 and
1.67, respectively. Panel B, NAD kinase assay. Pea NAD kinase
was partially purified and assayed as described under ``Materials
and Methods.'' The activity of NAD kinase is expressed as a
relative activity to that of NAD kinase in the presence of 80 nM SCaM-1. K
value of SCaM-1 for NAD kinase
was 8.07 nM and the Hill coefficient was
1.86.
Figure 7:
Expression of SCaM genes in
various tissues and organs of soybean. Panel A, expression of SCaM-1, -2, and -3 in various tissues of
4-day-old etiolated soybean seedlings. Twenty µg of total RNAs were
separated on 2% formaldehyde/agarose gels and transferred onto nylon
membranes. The blots were hybridized either with a P-labeled coding sequence probe or each SCaM gene-specific
probe as described under ``Materials and Methods.'' Tissues
examined were: A, apical hypocotyls; E, elongating
hypocotyls; M, mature hypocotyls; C, cotyledons; P, pulmules. Panel B, expression of SCaM-1, -2, and -3 in various organs of 5-week-old mature
soybean plants. Northern hybridizations were done as described in panel A. Organs examined were: YL, young leaves; OL, old leaves; ST, stems; RT, roots; SD, seeds; PD, pods; ND, nodules. Panel
C, expression of the SCaM-4 gene in soybean seedlings.
Northern blot analysis was performed with an SCaM-4-specific
probe using 2 µg of poly(A) RNA isolated from the apical (A), elongating (E), and mature (M) regions
of hypocotyls and roots (R) of 3-day-old etiolated seedlings. Panel D, RT-PCR analysis of SCaM-1 and SCaM-4 expression in seedlings. Total RNA (3 µg) were reverse
transcribed with an oligo(dT)
primer and subsequently PCR
amplified using isoform-specific oligonucleotide primers. Amplified
products were separated on a 2% agarose gel, hybridized with each cDNA
probe, and visualized by the ECL gene detection system. Closed arrow
heads (
) indicate 290-bp amplified cDNA copies of mRNAs encoding
SCaM-1 (left panel) and 380-bp amplified cDNA copies of mRNAs
encoding SCaM-4 (right panel), respectively. Internal
standards co-amplified for semiquantitive analysis are indicated by open arrow heads (
).
Figure 8:
Detection of SCaM-1 and -4 in soybean and
other plants by immunoblotting. Protein extracts from six different
plant species were prepared as described under ``Materials and
Methods.'' Sixty µg of total protein extracts in sample buffer
containing 5 mM CaCl were subjected to a 13.5%
SDS-polyacrylamide gel electrophoresis. Immunoblotting was done using
affinity-purified anti-SCaM-1 or -4-specific antibodies (for
preparation of isoform-specific antibodies, see ``Materials and
Methods''). Panel A, anti-SCaM-4 immunoblot. Panel
B, anti-SCaM-1 immunoblot. Size markers are indicated in the left
of panels A and B in kilodaltons. Lanes 1-6 contain soybean, Arabidopsis, chinese cabbage, rice,
tobacco, and tomato extracts, respectively. Lanes 7 and 8 contain 10 ng of purified SCaM-1 and SCaM-4,
respectively.
Calmodulin is one of the most highly conserved proteins in
higher eukaryotes. The essential role of calmodulin in a variety of
cellular processes may be the reason for strict conservation of the
primary structure of calmodulin during evolution. However, recent
studies in a plant system revealed the presence of multiple calmodulin
isoforms in a single
organism(4, 5, 6, 7) . This is very
interesting because there exists only a single form of calmodulin in
animal systems although calmodulin is encoded by multiple
genes(8, 9) . This implicates that the plant system
may have a unique feature although the overall
Ca/calmodulin-mediated signal transduction mechanism
is similar to that of animal systems. Plant calmodulin isoforms found
so far have minor amino acid sequence divergency such that the most
divergent plant calmodulin isoform, potato calmodulin, has the
difference of 10 amino acid residues from SCaM-1(34) . Most of
the other plant calmodulins have only one to six amino acid difference
and showed more than 90% identities at the amino acid sequence level.
In contrast to this, newly identified SCaM-4 and -5 have only 78%
identities to SCaM-1 and appear to be the most divergent calmodulin
isoforms found yet. Therefore, the study presented here not only
increased the number of calmodulin isoforms in plant systems but also,
more importantly, greatly extended the primary structural diversities
of functional calmodulins.
SCaM-4 had distinct characteristics
compared to the highly conserved SCaM-1 such as differences in the
extent of Ca-dependent electrophoretic shift,
antigenicity, and, most importantly, the ability to activate
calmodulin-dependent enzymes. To our surprise, SCaM-4 did not activate
NAD kinase at all although its ability to activate phosphodiesterase
was as good as SCaM-1. In addition, SCaM-4 did not inhibit the
activation of NAD kinase by SCaM-1 even in the presence of 100-fold
molar excess of SCaM-4 to that of SCaM-1, suggesting that SCaM-4 may
not bind NAD kinase at the assay condition used (data not shown). The
reason for this difference may be due to structural difference between
SCaM-1 and SCaM-4. Previous studies on the relationship between
structure and function of calmodulin by site-directed mutageneses or
isolation of mutants showed that only single critical amino acid
substitution could change the ability of calmodulin to activate target
enzymes(39, 40, 41) . Thus the failure of
SCaM-4 to activate NAD kinase is most likely due to the divergent
primary structure of SCaM-4. However, we cannot exclude the possibility
that plant phosphodiesterase is differently regulated by these
isoforms.
It is very intriguing to have such divergent calmodulin isoforms in plants compared to animal systems. Although, currently there is no definitive answers to the role(s) of the divergent isoforms, our studies with SCaM-1 and SCaM-4 provide important clues. The differential activation of calmodulin-dependent enzymes by different calmodulin isoforms suggests that each isoform may have its own target enzymes although some of them may be shared. This implicates that, in plant, there may exist calmodulin isoform-specific processes. However, this notion needs further studies on the identification of more calmodulin-dependent enzymes. Currently a very limited number of calmodulin-dependent enzymes have been identified or cloned in plants, which makes these studies difficult. Another possible function of plant calmodulin isoforms is signal-, tissue-, and/or developmental stage-specific roles. Consistent with this proposal, differential expression of two mungbean calmodulin isoforms in response to various stimuli have been reported recently(42) . Interestingly, the two mungbean calmodulin isoforms MBCaM-1 and MBCaM-2 have identical amino acid sequences to SCaM-1 and SCaM-2, respectively. Thus it is plausible that SCaM-1 and -2 may respond similarly to these various signals although SCaM-1, -2, and -3 showed the same expression patterns in various tissues we examined. Also the different expression level and patterns of SCaM-4 compared to other calmodulin genes support the notion of the tissue- and/or organ-specific functions of calmodulin isoforms. Further studies on the response of each calmodulin gene to various stimuli will clarify these possibilities.
Currently we are in the process of generating transgenic tobacco plants with sense and antisense constructs of SCaM-1 and -4 to investigate the biological function of each isoform in vivo. However, until now we were not very successful in obtaining transgenic plants. One of reasons may be due to the essential role(s) of calmodulin in various processes in cells, thus plants with perturbation of calmodulin expression level may not be able to survive.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L01430[GenBank]-L01433 [GenBank]and L19359[GenBank].