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Critical Reviews in Plant Sciences

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Calcium and Signal Transduction in Plants

a b c
B. W. Poovaiah , A. S. N. Reddy & Dr. Lewis Feldman
a
Department of Horticulture , Washington State University , Pullman, WA, USA
b
Department of Biology , Colorado State University , Fort Collins, CO, USA
c
Department of Plant Biology , University of California — Berkeley , Berkeley, CA, USA
Published online: 30 Mar 2011.

To cite this article: B. W. Poovaiah , A. S. N. Reddy & Dr. Lewis Feldman (1993) Calcium and Signal Transduction in Plants,
Critical Reviews in Plant Sciences, 12:3, 185-211

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 Critical Reviews in Plant Sciences, 12(3):185-211 (1993)

Calcium and Signal Transduction in Plants

B. W Poovaiah*
Department of Horticulture, Washington State University, Pullman, WA

A. S. N. Reddy
Department of Biology, Colorado State University, Fort Collins, CO

* To whom all correspondence should he addressed

Downloaded by [University of Illinois at Urbana-Champaign] at 13:00 10 March 2015

Referee: Dr. Lewis Feldman, Department of Plant Biology, university of California - Berkeley, Berkeley, CA

ABSTRACT: Environmental and hormonal signals control diverse physiological processes in plants. The mech-
anisms by which plant cells perceive and transduce these signals are poorly understood. Understanding bio-
chemical and molecular events involved in signal transduction pathways has become one of the most active
areas of plant research. Research during the last 15 years has established that Ca2+ acts as a messenger in
transducing external signals. The evidence in support of Ca2+as a messenger is unequivocal and fulfills all the
requirements of a messenger. The role of Ca2+ becomes even more important because it is the only messenger
known so far in plants. Since our last review on the Ca2+ messenger system in 1987, there has been tremendous
progress in elucidating various aspects of Caz+-signalingpathways in plants. These include demonstration of
signal-induced changes in cytosolic Ca2+,calmodulin and calmodulin-like proteins, identification of different
CaZ+channels, characterization of Ca2+-dependentprotein kinases (CDPKs) both at the biochemical and mo-
lecular levels, evidence for the presence of calmodulin-dependent protein kinases, and increased evidence in
support of the role of inositol phospholipids in the Ca2+-signalingsystem. Despite the progress in Ca2+research
in plants, it is still in its infancy and much more needs to be done to understand the precise mechanisms by
which Ca2+ regulates a wide variety of physiological processes. The purpose of this review is to summarize
some of these recent developments in Ca2+research as it relates to signal transduction in plants.

KEY WORDS: calcium, calcium channels, calmodulin, inositol phospholipids, signal transduction, signaling
pathways, messenger, protein kinases.

1. INTRODUCTION anisms in plants is in its infancy, considerable

progress has been made during the last 15 years
Environmental and hormonal stimuli affect in establishing Ca2+ as a messenger in signal
many aspects of growth and developmental pro- transduction. In the late 1970s, following the dis-
cesses in plants. Many of these signals evoke covery of calmodulin (CaM), a Ca2 -binding +

specific responses in plants. Because plant cells
respond to a great variety of stimuli, there must
be built-in mechanisms to sense and transduce
the signals. In recent years, understanding signal
transduction pathways - the series of biochem-
ical and molecular events that are involved in
evoking a final response - has become one of
the foremost areas of plant biology research. Al-
though understanding signal transduction mech-
protein, and CaM-dependent enzymes, it was
proposed that Ca2+ may act as a messenger in
signal transduction in plants. The realization that
Ca2+ may play a messenger role has resulted in
increased activity in Ca2+r e ~ e a r c h . ~Dur-
~*~~~,~~~
ing the past 5 years, there has been a dramatic
unfolding of information on the biochemical and
molecular aspects of the Ca2+messenger system
in p 1 a n t ~ . ~ It~ has , ~been
~ ~proposed
, ~ ~ that
~ ~ ~ ~ ~ , ~ ~

0735-2689/93/$. 50
0 1993 by CRC Press, Inc.

185
 Ca2+ should meet the following four criteria in
order to consider it to be a messenger in regu-
lating the physiological processes evoked by pri-
mary ~ t i m u l i : ~ ~ (1)
, ~ *cytosolic
~ ~ ~ * 'Ca2+
~ must
change in response to primary stimuli and such
change should precede the physiological re-
sponse; (2) artificial induction of changes in cy-
tosolic Ca2+ should evoke a physiological re-
sponse in the absence of primary stimuli; (3) cells
must possess the mechanism to sense the changes
in cytosolic Ca2+ and translate them into a phys-
iological response; and (4) blocking changes in
the cytosolic Ca2+ or Ca2+-sensingsystem must
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prevent the physiological response to external
stimuli. There has been considerable experimen-
tal evidence to satisfy criteria (2), (3), and (4);
however, evidence to support criterion (1) was
lacking until recently due to technical difficulties
in measuring changes in cytolosic Ca2+.63,75,145
Hence, the concept of a Ca2+ messenger system
in plants is widely prevalent but less substantiated
due to lack of evidence in support of signal-in-
duced changes in cytosolic Ca2+levels. In recent
years, tremendous progress has been made in
demonstrating signal-induced changes in the cy-
tosolic Ca2 level and in understanding the mode
+
of Ca2+ action. These studies indicate that Ca2+
is, probably, a universal messenger in transduc-
ing external stimuli in plants. Our goal here is
to review the recent developments in Ca2+ re-
search as it relates to signal transduction. Much
of the work that was covered in our earlier
reviewL45is not discussed here. For detailed dis-
cussions on Ca2 -mediated physiological re-
+
sponses and other aspects of Ca2+ involvement
in plant growth and development, the reader is
referred to other review^.^^^^^^'^^-'^^^^^^^^^'
II. EXTERNAL SIGNALS AND
CYTOSOLIC CALCIUM
A. Signal-Induced Changes in Cytosolic
Calcium
The concept that Ca2+ acts as a messenger
is based on the notion that environmental and
hormonal signals induce changes in cytosolic
Ca2+.Hence, determining the changes in the level
186
of cytosolic Ca2+ in response to external stimuli
has been crucial in establishing the role of Ca2+
as a messenger in plants. Although there was
circumstantial evidence that signals affect cyto-
solic Ca2+,it was not established until recently
by direct Ca2+ measurement in the cytosol. The
efforts to measure cytosolic Ca2+ in plant cells
using the methods that are routinely used in an-
imal systems have met with limited success. This
is due mainly to unique structural features of
plant cells such as the cell wall and the large
vacuole of plants and other problems such as dye
compartmentalization, leakage, and toxic-
ity. 30,63~145Use of Ca2 -binding fluorescent dyes
+
(quin-2, fura-2, and indo-1), Ca2+-selective elec-
trodes, and microinjection of aequorin have been
successfully ~ ~ e d . ~ ~These * ~methods
~ , ' ~have ' , ~ ~ ~
helped to establish that cytosolic Ca2+ in plant
cells is maintained in the range of 0.1 to 1 p M
and to demonstrate in some cases signal-induced
changes in cytosolic Ca2+.29,30,65,11X,119,145,177 Re-
cently, some novel approaches, such as using
transgenic plants that express a p o a e q ~ o r i ncon-
,~~
focal microscopy and fluorescence imag-
in38,37,59,67 and acid-loading of fluorescent dyes,30
have been developed that are successfully used
in monitoring the changes in cytosolic Ca2+lev-
els in response to various external stimuli.
Aequorin, a photoprotein, emits blue light
upon binding to Ca2+ ions. This protein, which
consists of apoprotein (apoaequorin) and a lu-
minophore (coelenterazine), has been used to de-
termine the cytosolic Ca2 concentration in some
+
cases.@s208 Aequorin is nonperturbing , nontoxic,
and has no problems of leakage and compart-
mentalization. Until recently, the only way to
introduce aequorin into cells was by microinjec-
tion. Hence, despite the advantages, the use of
aequorin was limited to large cells. Recently, the
Trewavas group developed a novel approach of
using aequorin to detect changes in cytosolic Ca2+
in plant^.^^.^^ This has opened up exciting new
possibilities for future Ca2+ research. Using re-
combinant DNA technology, they have been able
to generate transgenic plants that constitutively
express apoaequorin and reconstitute aequorin by
incubating the transgenic seedlings with coelen-
terazine. Transgenic plants that express apoae-
quorin are generated by transforming tobacco with
the coding region of apoaequorin complementary
DNA (cDNA) fused to cauliflower mosaic virus
 35s promoter. Using these transgenic plants,
Knight et a1.95,96have measured changes in cy-
tosolic Ca2+levels in intact plants in response to
various signals. Mechanical stimuli such as touch
and wind, and stress signals such as cold shock
and fungal elicitors, have been shown to induce
rapid and transient increases in cytosolic CaZ+
(Figure 1).95,96Based on the effect of ion channel
blockers on cold- and wind-induced changes in
cytosolic Ca2+ levels, it is suggested that the
source of Ca2+ is different for cold-shock and
wind signals.96Wind-induced cytosolic Ca2+in-
crease is unaffected by plasma membrane Ca2+
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channel blockers, whereas, it is abolished by or-
ganellar Ca2 channel blocker. Cold-induced cy-
+ tosolic Ca2+ levels and these methods are not
tosolic Ca2+ increase is blocked by plasma mem-
brane Ca2+ channel blockers and unaffected by
'1
U
a 1.5
Time
FIGURE 1. Signal-induced changes in cytosolic Ca2+
levels. Aequorin was reconstituted in transgenic plants
by incubating the tobacco seedlings expressing apoae-
quorin in a coelenterazine solution. These seedlings
were subjected to different signals, and the changes in
cytosolic Ca2+ were monitored by measuring lumi-
nescence. (a) The effect of touch-stimulus. Seedlings
were touched every 1 min with a fine wire (indicated
by arrows) after which cold water (0°C) was added.
(b) Seedlings were rapidly transferred from 20°C to dif-
ferent temperatures (0,5, 10, 20, and 40°C) by adding
water at the appropriate temperature. (From Knight,
M. R., Campbell, A. K., Smith, S. M., and Trewavas,
A. J., Nature, 352, 524, 1991. With permission.)
organellar Ca2 channel blockers. These results
+
indicate that the source for the increase in the
cytosolic Ca2+ by cold signal is likely to be ex-
tracellular and that for wind signal is intracel-
l ~ l a rThe . ~ movement
~ of tissue is a likely cause
in elevating cytosolic Ca2 by mechanical stimuli
+
such as wind and touch. Transgenic plants that
express apoaequorin have been very useful in
measuring relative changes in cytosolic Ca2 . +
However, quantification of the increased cyto-
solic Ca2+has not been done. Also, it is not clear
whether all cells or certain cell types respond to
the signals. Current methods for Ca2+ measure-
ments have been developed for determining cy-
applicable to monitor changes in the Ca2+ level
in cellular organelles. It is now possible to meas-
ure changes in cellular organelles by targeting
the apoaequorin to different organelles by fusing
apoaequorin to organelle-specific target se-
quences. Rizzuto et have targeted apoae-
quorin to mitochondria by fusing the mitochon-
drial presequences with the apoaequorin cDNA-
coding region and demonstrated agonist-induced
changes in mitochondria1 free Ca2+.
Stomata1 guard cells have been widely used
to establish the messenger role of Ca2+ and to
study Ca2+-mediated signaling pathways in
plants. 1 1 6 ~ 1 1The
9 effect of abscisic acid (ABA),
a growth regulator that controls stomata1 open-
ing, on the level of cytosolic Ca2+of guard cells
has been extensively i n ~ e s t i g a t e d . ~ ~ ~ ~ ~ ~ ' ~ ~ ~ ' ' ~ ~ ' ~
McAinsh et a1.'I8 were the first to demonstrate
an increase in cytosolic Ca2+ in guard cells in
response to ABA. However, in different studies,
the proportion of guard cells that showed an ABA-
induced increase in cytosolic Ca2+ varied be-
tween 37 and 80%, and the guard cells that did
not appear to show an increase in cytosolic Ca2+
were closed in response to ABA.66,86,118,179 Hence,
it was suggested that Ca2+-dependentand Ca2+-
independent ABA signal transduction pathways
may be operating in guard cells.'96 McAinsh et
al., ' l9 in their recent studies using fluorescence
ratio photometry and ratio imaging techniques,
concluded that the ABA-induced closure of sto-
mata is a Ca2+-dependent process and the in-
ability to detect an increase in a certain percent-
age (20%) of guard cells was attributed to
methodology.
187
 In protoplasts of maize roots, salt stress in-
creased cytosolic Ca2+.'I4 Using a scanning laser
confocal microscope, Gehring et al.59 demon-
strated rapid (3 to 5 min) light- and gravity-in-
duced changes in cytosolic Ca2+ during photo-
tropism and geotropism of maize coleoptiles.
Light-induced increase in cytosolic Ca2 was ob-
+
served in the cells on the shaded side of maize
coleoptiles and in horizontally placed coleoptiles
cytosolic Ca2+ was found to increase rapidly in
elongating cells on the lower side.59The observed
changes in the Ca2+ level were restricted to epi-
dermal and cortical cells. These results suggest
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that increased cell elongation stimulated by light
and gravity is correlated with changes in the level
of cytosolic Ca2+.In maize coleoptiles and par-
sley hypocotyls and their roots, auxin and ABA
were found to increase the cytosolic Ca2 within
+
4 min.60Gilroy and Jones,67using confocal mi-
croscopy and fluorescence ratio analysis, studied
the effect of gibberellic acid (GA) and ABA on
the cytoplasmic Ca2+ level in barley aleurone
protoplasts. Treatment of aleurone protoplasts
with GA increased cytosolic Ca2 concentration
+
threefold (50 to 150 nM). The increase in cyto-
solic Ca2+occurs after 4 to 6 h of treatment and
precedes GA-induced a-amylase synthesis and
secretion.67 Furthermore , GA-induced increase
in cytosolic Ca2+was confined to peripheral cy-
toplasm just inside the plasma membrane. The
GA-induced increase in cytosolic Ca2+ is prob-
ably due to Ca2+ influx from outside rather than
Ca2+ release from internal stores because low-
ering of the external Ca2+to 1 mM inhibited the
GA-induced Ca2+increase in the cytosol. ABA,
which blocks the GA-induced a-amylase synthe-
sis and secretion, reversed the GA-induced in-
crease in cytosolic Ca2+.Wang et a1.199also re-
ported that ABA induced a decrease in cytosolic
Ca2+ in barley aleurone protoplasts. However,
in stomata1 guard cells, maize coleoptiles, and
parsley hypocotyls, ABA increased cytosolic
Ca2+.60366,118,119
Hence, it is likely that the same
signal may have opposing effects depending on
cell type or tissue.
Taken together, these results provide direct
evidence that many of the environmental and hor-
monal signals (wind, touch, light, gravity, sal-
inity, auxin, ABA, and GA) induce rapid changes
in cytosolic Ca2+and support the hypothesis that
188
change in the cytosolic Ca2+ level is one of the
primary events in the transduction of many sig-
nals. From these studies, it is obvious that Ca2+
is likely a universal messenger in the transduction
of signals in plants.
In addition to signal-induced changes in cy-
tosolic Ca2+ , there are various instances where
Ca2+ gradients are observed within a cell and
localized increase in cytosolic Ca2 levels. Keith
+
et al.92have shown a localized transient increase
in free Ca2+ at the mitotic spindle pole during
anaphase of mitotic endosperm cells of Huemun-
thus. A similar localized increase in free Ca2+
was observed during anaphase in stamen hair cells
of T r u d e s ~ u n t i uand
~ ~ other cell types in ani-
mals. 157 Using Ca2 -selective electrodes and a
+
fluorescent Ca2 indicator, a longitudinal gra-
+
dient of cytosolic Ca2+ was shown in growing
rhizoid cells of Fucus s e ~ r u f u s The . ~ ~gradient
~~~
of Ca2+ appears to be maintained by a prefer-
ential influx of Ca2+into the growing tip region.
In growing pollen tubes of Lillium longiglorum,
a continuous gradient of free Ca2+ is observed.
The pollen tube tip contained about five times
higher free Ca2+ as compared to the pollen tube
base. 130 It is likely that signal-induced changes
also may be confined to localized regions in the
cytoplasm. For instance, a GA-induced Ca2+in-
crease in barley aleurone cells is observed in pe-
ripheral cytoplasm.67
Recent studies indicate that Ca2 regulates
+
gene expression in both plants and animals (see
Section IV). However, it is not yet known as to
what extent Ca2+ fluctuations in the cytosolic
Ca2+ level are reflected in changes in nuclear
free Ca2+ concentration. It is believed that the
ions and small molecules diffuse in and out of
the nucleus freely through the nuclear pores.
However, recent studies show that there are Ca2+
gradients between the cytoplasm and the nu-
c l e u ~ indicating
, ~ ~ ~ that there could be mecha-
nisms that regulate Ca2+ transport into and out
of the nucleus. Nicotera et a1.128have shown that
ATP stimulated Ca2+ uptake and increased the
intranuclear free Ca2 concentration in rat liver
+
nuclei, suggesting the presence of an active Ca2+
uptake system in the nuclei. In addition, the role
of CaM also is implicated in ATP-stimulated Ca2+
uptake by the nuclei. More recently, Birch et a1.I4
demonstrated intranuclear Ca2 transients during
+
 neurite regeneration. Furthermore, using the
patch-clamp technique, Mazzanti et al. 'I7 showed
that the nuclear envelope contains ion channels.
At the present time, no information is available
on the level of free Ca2+ in the plant nuclei or
if there is a gradient between the cytoplasm and
nucleus.
B. lnositol Phospholipids and Calcium
Channels
There is now overwhelming direct evidence
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that many signals induce changes in cytosolic
Ca2+. However, the mechanisms by which sig-
nals cause changes in cytosolic Ca2+ are poorly
understood at the present time. Depending on the
signal and on the cell type, the cytosolic Ca2+
level could increase by influx of extracellular
Ca2+,by release of Ca2+ from internal stores,
or a combination of both. Studies with plant cells
suggest that the influx of Ca2+ at the plasma
membrane level and/or release from intracellular
stores, especially from vacuoles, are involved in
the p r o c e s ~ . It~is~likely
~ ~ ~that
~ different
' ~ ~ sig-
nals may use different mechanisms to bring about
changes in cytosolic Ca2+. For instance, using
Ca2+ channel blockers specific to plasma mem-
brane and organelles, Knight et al.96concluded
that cold-induced increase in cytosolic Ca2+ is
due to an influx of Ca2 , whereas wind-induced
+
increase is due to Ca2+ release from internal
stores. External Ca2+ is the likely source in the
case of GA-induced increase in cytosolic Ca2+
in barley aleurone pro top last^.^^ ABA-induced
changes in cytosolic Ca2 have been attributed
+
to both Ca2+ influx and Ca2+ release from in-
ternal stores. 119~179
The role of phosphoinositides,
Ca2+ channels, and Ca2+ ATPases in elevating
cytosolic Ca2+ in response to signals as well as
in Ca2+ homeostasis in plant cells is discussed
below.
In animal cells , phospholipase C-mediated
hydrolysis of phosphatidylinositol-4,5-bisphos-
phate (PIP,) plays a crucial role in elevating cy-
tosolic Ca2 in response to signals and in Ca2 -
+ +
signaling pathways. l 2 Signals through receptors
and G-proteins activate phospholipase C. The ac-
tivated phospholipase C catalyzes the hydrolysis
of PIP, into inositol- 1,4,5-trisphosphate (IP,) and
diacylglycerol (DG), which play a key role in
signal transduction.l 2 IP, elevates cytosolic Ca2+
by releasing Ca2+from internal stores, especially
from the endoplasmic reticulum (ER); and DG
activates protein kinase C, a Ca2+- and phos-
pholipid-dependent protein kinase, which in turn
regulates the activity of several enzymes and pro-
teins by pho~phorylation.~,'~~ Studies from var-
ious laboratories indicate the presence of many
of the components of PIP, pathway and their
possible role in signal transduction in plants (re-
viewed in References 20, 49, 85, 103, 124, and
145). The presence of polyphosphoinositides,
kinases involved in the synthesis of PIP and PIP,,
and phospholipase C has been shown in different
plant ~ y ~ t e m ~ . Hydrolysis ~ ~ . ~ of ~ PIP,
~ , ~ ~ ~ , ~ ~
has been shown by light and a u ~ i n . ' ~IP~ , one
37'~~
of the hydrolysis products of PIP2, has been shown
to release Ca2+from vacuoles isolated from var-
ious t i s s ~ e s ' ~ ~by
, ' ~activating
' Ca2+ channels. '
Blatt et al.I5 showed reversible inactivation of
K + channels by IP, in guard cells. Gilroy et al.65
demonstrated that IP, elevates Ca2 in guard cells
+
and elicits stomatal closure. IP,-gated Ca2+chan-
nels of vacuoles showed several properties that
are similar to the animal IP,-gated channel lo-
cated on ER.24There is no conclusive evidence
for the presence of protein kinase C, which is
activated by Ca2+ and DG. However, DG en-
hanced light-induced stomatal opening in Com-
melina communis and induced stomatal opening
under darkness.'02 It was shown by whole-cell
patch clamping of guard cell protoplasts that DG
induce ion pumping. Taken together, these re-
sults suggest that IP, is clearly involved in ele-
vating cytosolic Ca2+, although the source of
Ca2+, unlike in animals, is the vacuole and DG
is involved in regulating channels, at least in
guard cells. However, additional work is clearly
needed to establish the role of DG in stimulus-
response coupling. Studies on G-proteins, which
are known to be involved in the activation of
phospholipase C as well as in regulating ion chan-
nels in animal cells, have just begun in ~ l a n t s . ~ ~ , ' ~ '
The presence of G-proteins has been shown in
plants, and some of the genes that code for these
proteins have been cloned from plants.
18375,76~115~137
Recently, Fairley-Grenot and Assmann5' pro-
vided evidence that G-proteins in guard cells of
fava bean regulate K + channels in the plasma
189
 membrane. These results indicate that in plants,
as in animals, regulation of ion channels by en-
vironmental stimuli may be mediated by G-pro-
teins.
The existing evidence suggests that Ca2+
channels, which are responsible for influx of Ca2
from extra- and intracellular compartments, play
a key role in elevating cytosolic Ca2+ in plant
cells in response to signals.90,178 Relatively little
is known about Ca2+channels in plants as com-
pared to animal systems. In animal systems, dif-
ferent types of Ca2+ channels have been char-
acterized. These include voltage-dependent,
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ligand-regulated, and stretch-activated Ca2+
channels.88There are four classes of voltage-sen-
sitive channels (L, N, P, and T classes) of which
L, N, and P type require a large amount of de-
polarization before they are activated, whereas T
(transient)-classchannels are activated with small
amounts of depolarization and inactivated by large
amounts of depolarization.82*88 L, N, and P chan-
nels differ in their sensitivity to drugs such as
1,4-dihydropyridinechannel blockers and w-con-
toxin GVIA (w-CgTX). L channels are sensitive
to 1,4-dihydropyridines and insensitive to w-
CgTX, whereas N channels are insensitive to 1,4-
dihydropyridines but are blocked by o-CgTX. P
channels are insensitive to both 1,6dihydropyr-
idines and o-CgTX.88
Recent studies in plants indicate that there
are several types of Ca2+ channels in plasma
membrane and vacuole that are regulated by dif-
ferent mechanism^.^.^^^ Voltage-dependent Ca2
channels have been detected on the plasma mem-
branes of algae.90v185 Studies with drugs that block
Ca2+ channels indicate the presenct of voltage-
dependent Ca2+channels in higher plants.goDur-
ing ABA-induced stomata1 closure, cytosolic
Ca2+ in guard cell increases. Schroeder and
H a g i ~ a r ahave
' ~ ~ shown that ABA activates non-
selective Ca2+permeable channels on the plasma
membrane of guard cells. There also is evidence
for the presence of stretch-activated Ca2 chan- +
nels on the plasma membrane .42,46 Intracellular
organelles act as Ca2+ stores, and the opening
of Ca2+channels on these organelles contributes
to elevation of cytosolic Ca2+. Two classes of
channels have been identified on the vacuolar
membrane.% One type of Ca2+ channel in the
vacuole is activated by IP,. 1 ~ 1 5 s ~ 1 In
8 1 animal cells,
190
IP,-gated Ca2+ channels are found on ER.l2 A
second tonoplast Ca2+ channel that is voltage-
gated and IP,-insensitive has been identified.90J38
The tonoplast voltage-dependent Ca2 channels+
have several properties comparable to animal L-
+ type channels. The chloroplast envelope mem-
brane appears to have Ca2 +
So far, no Ca2+ channels have been reported in
the ER of plant cells and other organelles such
as mitochondria. Virtually nothing is known about
the biochemical properties of Ca2+channels and
the mechanisms involved in regulation. Mem-
brane proteins that bind to Ca2 channel blockers
+
with high affinity have been identified and in
some cases solubilized and partially puri-
fied.4,68,74,1 9 0 1nformation on Ca2+ channels in
plants is beginning to be accumulated. Future
studies using electrophysiological, biochemical,
and molecular techniques on Ca2+channels will
undoubtedly contribute to identification of new
Ca2+ channels and the mode of regulation of
different Ca2+channels by signals, which is vital
for our understanding of how signals induce
changes in cytosolic Ca2+.
Regulation of low cytosolic Ca2+ levels de-
pends on the concerted operation of Ca2+-pump-
ing systems located on the plasma membrane and
intracellular organelles. In plants, Ca2 -ATPases
+
localized in the plasma membrane and ER have
been identified by Ca2 transport studies
+ In
.50384
addition, tonoplast-bound H /Ca2 antiport also
+ +
would reduce cytosolic Ca2 by sequestering it
+
+
into the vacuole.180~181Recently, Wimmers et a1.209
isolated and characterized a cDNA that codes for
putative ER-localized Ca2 -ATPase in tomato.
+
A cDNA sequence that codes for a partial se-
quence of ER-Ca2 -ATPase was isolated from
+
tobacco. 142 The plant Ca2 -ATPase localized in
+
the ER has about 50% amino acid sequence iden-
tity with animal ER-ATPases. The expression of
the ER-Ca2+-ATPase was found to be induced
by salt stress.
111. CALCIUM-MODULATED PROTEINS
The increase in free Ca2+concentration in a
cell has generally been portrayed as a switch turn-
ing on various cellular processes. However, in
most cases, the changes in cytosolic Ca2+ are
 sensed by a group of Ca2+-modulated proteins conveys the signal from the cell surface to the
that are believed to be involved in the regulation metabolic machinery, eventually resulting in a
of many cellular activities. These binding pro- physiological response. So far, two Ca2 -binding +

teins have a structural feature called EF-hand, proteins have been well characterized in plants.
which is present in multiple copies and binds CaM, a well-characterized Ca2 -binding protein
+

Ca2+ with high affinity and undergoes a confor-
mational change leading to a change in their abil-
ity to interact with other proteins or a change in
their a~tivity.~'A large number of Ca2+-binding
present in all plants and animals, is believed to
play a key role in Ca2+-signalingpathways in
plants. A second Ca2+-bindingprotein called
Ca2 -dependent and CaM-independent protein
+

proteins have been identified and characterized
in animals and a few in plant^.^^,^^^,^^^ Ca2+-
binding proteins are inactive in the absence of
bound Ca2+.When the concentration of cytosolic
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kinase, which is unique to plant systems, also is
fairly well characterized.170 In addition, there are
indications for the presence of CaM-like proteins
and additional Ca2 -binding proteins that could
+

Ca2 increases, the Ca2 -binding proteins, such

+ + be involved in sensing cytosolic Ca2+ changes
as CaM and Ca2 -dependent protein kinases bind
+
and mediating Ca2 action in plants .21,36,98,212
+

to Ca2+. Upon binding to Ca2+,these proteins

become active and interact with other proteins in
the cell and alter their activity. Figure 2 shows
a schematic diagram that illustrates how elevated
levels of cytosolic Ca2+ affect various enzymes
and proteins in the cell. Hence, in the chain of
events, Ca2+ acts as a simple on-off switch that
A. Calmodulin
The presence of CaM in plants was first re-
ported in the late 1970s.2,3,126
CaM has since been
purified and characterized from a number of plant

C Y ~ O S O ~Caz+
C expression of
Regulation
~~ of C ~ M ression of
Regulation
and CaM-like proteins C%-binding proteins

Activation of Ca2+- Ca2+-CaM

dependent enzymes
(e.g., CDPKs) I
CaM-binding proteins
(e.g., protein kinase, ATPases)

RESPONSE
FIGURE 2. Schematic illustration of the proposed events involving Ca2+,CaM, and CBP
in signal transduction. Signals induce changes in cytosolic Ca2+ and these changes in
cytosolic Ca2+ are transmitted to the metabolic machinery through CBP. The proposed
events are based on existing experimental evidence.21.22.30.5'6~.73.89.95.96.111,118.119,145.150.162

191
 systems.168CaM is a highly conserved protein
and is considered to be a multifunctional protein
because of its ability to interact and regulate the
activity of a number of other proteins. Biochem-
ical and physical properties of plant CaM are very
similar to animal CaM. These and other aspects
of CaM are reviewed in Roberts et a1.168and
Poovaiah and R e d d ~ , hence
' ~ ~ they are not dis-
cussed here. During the last 4 years, there has
been considerable progress in studying CaM gene
expression and the organization of CaM genes in
plants. Much of our focus here is on the recent
developments in CaM research.
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1. Calmodulin Gene Structure and

Expression

The amino acid sequence of spinach and

wheat CaM was first obtained by protein se-
quencing. 112,193 cDNAs or genomic clones that
code for CaM were isolated from Chlumydu-
m u n ~ s potato;89
; ~ ~ ~ barley;'07,212Arubidup-
sis;21,108,140,212 petunia;35alfalfa;'O tomato;38 rice;34
potato
and apple.202 Alignment of amino acid sequences Tomato
Petunia
of different plant CaM and CaM-like proteins Petunla
Patunla can53
with that of an animal CaM is shown in Figure Rice
RIce
Rice can1
3. CaM from different plant systems have a high 0drl.y
A 1 fa1 fa
degree (92% or more) of amino acid sequence Carrot
At can2
conservation among themselves and between plant At can4
At can6
Spinach
and animals (Figure 3). Although plant CaM have wheat
Chlanydomonas
some unique properties, they share a high degree Rat
TCH2
of functional conservation with animal CaM. 168 TCH3
CDPK(Corn) Y-TVD--WIIACKEHNMPMTL-DV-K---Q-N--RID-C--AET
Among all the CaM, the amino acid sequence of CDPK (Soybean) Y - T L D - I W 1 I A C K D F G - D D I H I - D - - - - - I - Q - N - - I D - G
CDPK (Carrot1 Y-TKD--ESll~II(T~GIICD-rTlKV-KSE-V-S--RID-N-D-PCM
yeast shows significant sequence divergence (60%
identity with other CaM).44Deletion of CaM gene FIGURE 3. Amino acid sequence comparison of CaM
in yeast is lethal. However, overexpression of from plants and animals. Amino acids that are believed
vertebrate CaM in yeast cells lacking the CaM to be involvedin Ca2+-bindingare denoted by asterisks.
Hyphens indicate the identities in amino acid, and #
gene results in normal growth of yeast, suggest- indicates no amino acid present at that position. Al-
ing that yeast and vertebrate CaM are functionally falfa;'O Arabidopsis CaM-2, CaM-4, and CaM-6;58.10e
similar despite significant structural differ- barley;lo7carr~t;'~' Chlamyd~monas;"~ petunia;35po-
ences."~'~~ Isolation of cDNA and genomic clones tat^;^^ tomato;38 ice;^^,'^ wheat;i93 spinach;'" rat.184
has helped to determine the number and orga- Deduced amino acid sequences of CaM-like proteins
nization of CaM genes and to analyze the expres- such as TCH2 and TCH3 from Arabidopsis2' and Ca2+-
binding region of Ca2+-dependentprotein kinases are
sion of CaM genes. from and corn.13
Recent studies have shown that CaM and
CaM-related genes are highly responsive to sig- gene expression in auxin-responsive strawberry
nals. Various physical and chemical signals have fruit'61 and light-responsive Merit corn roots.
been shown to induce mRNA corresponding to Seven-day-old fruits were depleted of auxin by
CaM and CaM-related genes. Using potato CaM deachening and treated with or without auxin and
cDNA as a probe, Jena et al.89investigated the the RNA isolated from these fruits was probed
effect of signals such as auxin and light on CaM with CaM. A higher level of CaM message is
192
 found in auxin-treated strawberry fruits as com-
pared to Exposure of dark-grown Merit
corn root tips to light also increased CaM mRNA
level. In Arubidopsis, Braam and Davis2]showed
a rapid (10 to 30 min) induction of mRNAs cor-
responding to four cDNAs (TCH 1, TCH 2, TCH
3, and TCH 4) in response to a variety of stimuli
such as touch, wind, rain, and wounding. Of
these four genes, TCH 1 is identified as CaM
and TCH 2 and TCH 3 are identified as CaM-
related genes. In apple, the expression of CaM
is induced by wounding.202 These studies suggest
that physical and chemical signals induce the
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expression of CaM. By manipulating cytosolic
Ca2+,the expression of some of the touch genes
is found to be regulated by Ca2+.22 Recent studies
have shown that touch and wind signals elevate
cytosolic Ca2+.95,96 Hence, the probable se-
quence of events in touch signal transduction is
elevation of cytosolic Ca2+,which in turn reg-
ulates the expression of the specific genes, in-
cluding those that code for its own receptor. Re-
cent studies indicate that Arubidopsis and other
higher plants contain multiple genes that code for
CaM. In Arubidopsis, there seems to
140~1503162*212
be multiple CaM genes (ACaM-1, ACaM-2, and
ACaM-3) and the expression of the three genes
was found to be differentially regulated. 108~140For
instance, in roots, mRNA corresponding to only
ACaM-1 was detected, whereas ACaM-2 and
ACaM-3 are not expressed. Hence, it is likely
that different CaM genes may have different reg-
ulatory elements. Although the nucleotide se-
quence of ACaM- 1, -2, and -3 is diverged (about
86% identity), ACaM-2 and ACaM-3 genes code
for identical proteins, whereas the protein coded
by ACaM-1 differed from ACaM-2 and -3 by
four conservative substitutions outside the Ca2 -
+
binding domains. In humans, there are three dis-
tinct CaM genes that code for identical
The amino acid sequence of TCH-1 is identical
to ACaM-2 and -3. All the three known CaM
genes in Arubidopsis are induced by touch stim-
ulus, although the kinetics and the fold induction
varied between different genes. 140 In Arubidopsis
ACuM-3, the coding region is interrupted after
the 25th amino acid by a single 491 bp intron.
In rice, CaM also is coded by a multigene family.
Two of the rice CaM genes (CaM-1 and CaM-
2) have been cloned and characterized.150,162 Us-
ing Northern analysis, both the genes were found
to be expressed. The deduced amino acid se-
quence of rice CaM-1 and CaM-2 is identical,
except for one replacement at the 139th amino
acid. Both of the rice CaM genes have a single
intron (1400 bp long in CaM-1 and 770 bp long
in CaM-2), which disrupts the coding region at
the 25th amino acid. In apple, the CaM-coding
sequence is interrupted by a single intron (860
bp) at the 25th amino acid.202Hence, the number
and position of the intron seem to be conserved
in higher plants. In Chlumydomonus, CaM is
coded by single gene with five introns and the
position of the first intron is the same as Aru-
bidopsis, rice, and apple.213Vertebrate CaM genes
have several introns and the intron positions show
considerable c ~ n s e r v a t i o n However,
.~~ none of
the vertebrate CaM genes have an intron at the
25th amino acid.
The availability of CaM genomic clones from
Arubidopsis, rice,34 and apple202will help in
identifying regulatory elements in CaM pro-
moters. To study the regulation of rice CaM gene
expression, we have generated transgenic plants
carrying a chimeric fusion between the rice CaM-
2 promoter and CAT reporter gene, and the ac-
tivity of CaM-2 promoter is determined by as-
saying CAT activity. The reporter gene is ex-
pressed in all the vegetative and reproductive
organs tested, suggesting that the promoter is
active in all plant organs. However, higher CAT
activity was observed in stem, petioles, and vein
tissues, and much lower activity was detected in
leaf blade. Also, different parts of younger leaves
showed higher activity as compared to older
leaves. Among the various parts tested, anthers
showed the highest promoter activity (Figure 4).
Heat shock reduced CAT activity, whereas 2%
sugar in the incubation medium increased CAT
activity.163
In addition to CaM, there are reports of CaM-
related genes in plant^.^'.^^,^^^ In A rubidopsis, a
cDNA for a CaM-like protein (p21) that shares
65% amino acid similarity with the higher plant
CaM sequences has been isolated. 110*212From the
same system, Braam and Davis21 isolated two
partial cDNAs, TCH 2 and TCH 3, that code for
CaM-related proteins, which showed 4-4and 70%
amino acid identities, respectively, with CaM.
The p21 has several unique structural features,
including a 45-amino acid carboxy-terminal ex-
tension with no homology to any known proteins.
193
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S P C A
FIGURE 4. Rice CaM-2 promoter activity in different parts
of flower in transgenic plants. S, sepals; P, petals; C, carpels;
A, anthers. Transgenic plants containing CaM-2 promoter
fused to chloramphenicol acetyltransferase (CAT) reporter
gene were obtained and the CaM-2 promoter-driven CAT
activity was measured in different parts of the plant. Equal
amounts (40 p.g) of protein were used for assaying CAT ac-
tivity.'B3

Another CaM-related cDNA was isolated from
petunia, which contained an extra domain of 35
amino acids at the carboxy-terminal end. Of the
amino acid residues in the extra domain of pe-
tunia CaM-like protein, 40% is positively
charged.35The significance and function of these
CaM-like proteins are not known at this time.
2. Manipulation of Calmodulin Levels in
Transgenic Plants
The role of CaM has been implicated in con-
trolling a number of physiological processes and
much of this is based on the use of CaM antag-
o n i s t ~ . ~ In ~yeast,
* ~ CaM
~ ~ *is~essential
~ ~ for cell
proliferation because deletion of the gene encod-
ing CaM is letha1.44.214 Recent advances in mo-
lecular techniques have offered new approaches
to understand the role of CaM in plant growth
and development by overexpressing or blocking
the expression of CaM. Transient overproduction
of CaM levels in transformed mouse cells ac-
celerated cell proliferation. Furthermore, de-
creased CaM levels by antisense RNA resulted
in the arrest of cell c ~ c 1 e . lHowever,
~~ over-
production of CaM (up to 100-fold as compared
to normal level) in yeast did not affect any growth
or other characteristics in yeast.45To study the
consequences of altered levels of CaM on plant
growth and development, we regenerated several
independent transgenic tobacco plants containing

194
 potato CaM cDNA in a sense or antisense ori-
entation driven by the CaMV 35s promoter.
Characterization of these plants revealed up to
50-fold higher levels of the CaM mRNA in plants
containing the sense constructs, whereas plants
carrying the long construct in antisense orienta-
tion showed generally less of the CaM message
when compared to the controls. 150,162 In addition,
the steady-state level of the CaM antisense mRNA
was much lower compared to the CaM mRNA
driven by the CaMV 35s promoter. However,
the amount of the CaM protein was not signifi-
cantly affected, as judged by CaM activity and
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Western blotting, in these transgenic plants as
compared to the control. These results suggest
that the amount of CaM in the plant cell could
be regulated stringently either at the translational
and/or post-translational level. It also is possible
that CaM mRNA derived from cDNA may not
be translated or the translated protein may be
degraded because of the heterologous system.
However, this possibility is unlikely inasmuch as
tobacco has been used to obtain transgenic plants
containing heterologous cDNAs driven by CaMV
35s promoter, and the introduced foreign genes,
including animal genes, were properly expressed
both at the mRNA and protein level in a number
of instance^.^^,^^ Furthermore, potato and tobacco
are closely related phylogenetically and belong
to the same family. This seems to be unique to
plants because a 5- to 80-fold increase in the CaM
protein was achieved in transformed yeast and
mammalian s y ~ t e m s . ~R,~ plants
* ' ~ ~ that we ob-
tained showed no significant phenotypic changes.
Roberts et al. 169 also reported no changes in phe-
notype of transgenic plants expressing a twofold
higher level of CaM as compared to controls.
Zielinski et a1.212also have generated transgenic
plants carrying barley CaM cDNA fused to the
CaMV 35s promoter in sense and antisense ori-
entations. Plants containing sense constructs
showed significantly higher CaM mRNA and the
plants with antisense constructs showed reduced
level of CaM mRNA as compared to control.
However, very few differences were observed in
CaM protein between transgenic and control
plants. Although the R, plants appeared mor-
phologically normal, after germination of seeds
obtained by selfing & plants containing sense
construct, about 10 to 25% of the seedlings
showed morphological abnormalities such as short
internode length and lack of apical dominance.212
However, it is not clear if these observed ab-
normalities are due to increased expression of
CaM.
CaM undergoes post-translational methyla-
tion at position 115, which is catalyzed by a
specific CaM-N-methyltransferasc.173 Methyl-
ated and unmethylated CaM have similar prop-
erties with regard to activation of target enzymes
except in the case of activation of NAD kinase.
Also, trimethylation of CaM is believed to protect
CaM from degradation by the ubiquitin path-
way.69Recently, Ca2+-dependent polyubiquitin-
ation of plant CaM at a single site was demon-
strated.211Methylation of CaM reduces the
activation of NAD kinase by about threefold. 168
In pea plants, the level of CaM methylation var-
ied depending on the developmental state of the
tissue.'34 Changes in the levels of CaM also are
observed during different stages of carrot cell
cultures, indicating that methylation of CaM is
regulated by the stage of cell growth. Recently,
Roberts et al. 169 addressed the functional signif-
icance of CaM methylation by generating trans-
genic plants expressing normal (VU-1) and meth-
ylation mutant (VU-3) CaM. VU-1 and VU-3 are
identical except that VU-3 cannot be methylated.
Introduction of these foreign CaM resulted in a
twofold increase in the CaM level, and the amount
of foreign CaM was found to be equivalent to
that of endogenous CaM. Transgenic plants ex-
pressing CaM methylation mutant (W-3) showed
decreased stem internode, reduced seed and pol-
len viability, and reduced seed production. How-
ever, the transformants expressing VU- l were
found to be indistinguishablefrom control plants.
The observed differences in the transgenic plants
containing VU-3 have been attributed to mutant
CaM .
3. Calmodulin-Dependent Enzymes and
Other Calmodulin-Binding Proteins
A transient increase in cytosolic Ca2+ is
transmitted to the metabolic machinery through
Ca2+-binding protein^.^^,^^.'^^ CaM, upon bind-
ing to Ca2+,interacts with a number of key en-
zymes and other structural proteins called CaM-
binding proteins (CBP), which play a key role in
cellular r e g ~ l a t i o n . ~ ~CaM
, ' * ~ is unique because
195
 of its ability to interact with and control the ac-
tivity of many target proteins. A number of CBP
have been isolated, characterized, and identified
in These include protein kinases,
protein phosphatase (calcineurin), nitric oxide
synthase, Ca2+ ATPase, IP, kinase, and several
structural protein^.^^,^^ This has greatly increased
our understanding of how Ca2 and CaM regulate
+
the various biochemical and molecular processes
that eventually lead to a physiological response.
Very little is known about the number, locali-
zation, and identity of CBP in plants, although
enzymes such as NAD kinase, Ca2+ ATPase,
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nuclear NTPases, and protein kinases are known
to be activated by CaM.145,170 The lack of infor-
mation on the CBP and their identities has been
a major limitation in elucidating the Ca2 -me-
+ are known to inhibit CaM-independent enzymes,
diated signal transduction mechanisms in plants.
Because the regulation of the enzymes - NAD
kinase, Ca2 -ATPase, and NTPases - has been
+ cation of CaM-dependent protein kinase is needed
discussed thoroughly in recent review^,'^^,^^^ it
is not covered here. Instead, we would like to
review the evidence in support of CaM-depen-
dent protein kinases in plants.
Protein phosphorylation, which is catalyzed
by protein kinases, is one of the major mecha-
nisms of signal integration in eukaryotic cells.
Protein kinases play a pivotal role in a majority
of the signal transduction
Extracellular signals, either directly or through
second messengers, regulate the activity of pro-
tein kinases, which in turn regulate the activity
of their substrates by phosphorylation. The di-
verse actions of various signals and amplification
of signals are largely achieved through protein
kinases.40 Extensive studies in animal systems
indicate that CaM-dependent protein kinases are
central to Ca2 -mediated signal transduction
+ different tissues of corn, Arubidopsis, and pea
pathways.41Five types of CAM-dependent pro-
tein kinases (CaM kinase I, CaM kinase 11, CaM
b a s e m,phosphorylase kinase, and myosin light
chain kinase) have been well characterized in
mammalian systems, although there are other
CaM-dependent kinases in animals.41,57,93 All
these CaM-dependent protein kinases, except
CaM KII, have limited substrate specificity. CaM
KII phosphorylates a wide range of substrates;
hence, it is considered to be a multifunctional
protein kinase. CaM KIT is present in different
species of vertebrates, invertebrates, yeast, and
196
other f ~ n g i . ~cDNAs
~ , ' ~ that
~ code for five dif-
ferent polypeptides (a,p, p', y, and 6) of CaM
KII have been isolated and characterized.192 Be-
cause of the important role played by CaM kin-
ases in animals, plant scientists have attempted
to identify CaM-dependent protein kinases. Sev-
eral reports have indicated the presence of CaM-
dependent protein kinases (reviewed in Refer-
ences 145 and 168). A majority of these studies
have been performed with crude protein prepa-
rations and the conclusions drawn were based on
inhibition of Ca2 -stimulated protein phosphor-
+
ylation by CaM inhibitors or stimulation of phos-
phorylation by exogenous CaM. Many of these
studies are considered inconclusive because of
(1) the nonspecific effects of CaM inhibitors that
and (2) the high concentration of CaM required
to stimulate phosphorylation. Although purifi-
to establish unequivocally the presence of CaM-
dependent enzymes, some of the recent results
coupled with earlier r e p o ~ t s ' ~ , ' ~strongly
,~~,~~~,'~~
suggest the presence of CaM-dependent protein
kinases in plants.
The presence of CaM-dependent protein ki-
nase in plants was investigated using affinity pur-
ified antipeptide antibodies produced against the
a-subunit of rat brain CaM KIT and specific sub-
strates for CaM KII.1509162 We used affinity pur-
ified antipeptide antibodies (RU- 16) to rat brain
CaM KII a-subunit. Antibody RU-16 was pro-
duced against the synthetic peptide correspond-
ing to amino acid residues 378-402, which are
located in the regulatory domain of CaM KIT.
This antibody recognizes all isoforms of CaM
KII. Soluble and membrane proteins isolated from
were probed with RU-16. As shown in Figure 5,
RU-16 detected one or two bands (54 to 56 kDa)
in soluble proteins. The molecular weight of the
cross-reacting proteins is similar to the mam-
malian CaM KII. Two other antipeptide antibod-
ies (G-250 and G-301 raised to synthetic peptides
corresponding to 28 1-291 and 28 1-302 amino acid
residues of the a-subunit) recognized the same
bands. The synthetic peptide that is used for rais-
ing antibodies is not from the catalytic domain
of CaM KII and is unique to CaM KII; hence,
the argument that the RU- 16 antibodies may cross
 FIGURE 5. lmmunoblotting of proteins from various plant tissues with anti-
peptide antibodies to the rat brain CaM KII. Soluble proteins from different
Downloaded by [University of Illinois at Urbana-Champaign] at 13:00 10 March 2015

plant tissues were probed with RU-16. Lane 1: Merit corn root base; lane 2:
merit corn root tip; lane 3: pea root base; lane 4: pea root tip; lane 5: pea
stem base; lane 6: pea stem elongation zone; lane 7: pea stem tip; lane 8:
Arabidopsis root; lane 9: Arabidopsis leaf; lane 10: Arabidopsis stem; lane
11 : Arabidopsis silique; and lane 12: Arabidopsis

react with other protein kinases because of the new avenues in Ca2 research. Northern analysis
+

homology in the catalytic domains of protein kin-
ases is ruled out. In addition, a soluble extract
from corn roots phosphorylated synapsin I, a
physiological substrate for mammalian CaM KII,
and this phosphorylation was stimulated by Ca2+
and CaM. Phosphopeptide mapping has indicated
that the site of enhanced phosphorylation of syn-
apsin by a plant protein kinase is located within
the 30-kDa fragment, which is the known site of
phosphorylation by CaM KII. BB40, a specific
peptide substrate corresponding to residues 28 1-
291 of the a-subunit of CaM KII, also is phos-
phorylated by plant extract in a Ca2+ -CaM-de-
pendent manner. These results suggest that plants
contain a homolog of CaM KII. Recently, Wa-
tillon et aLZo1isolated a cDNA (CB1) clone by
screening an expression library with 1251-labeled
CaM. The deduced amino acid sequence of this
CBP showed sequence similarities with rat brain
CaM KII isoforms. The similaritiesinclude CaM-
binding domain and domain XI of protein kinase,
suggesting that the plant CBP could be CaM ki-
nase (Figure 6). The missing 3' and 5' ends of
the CBl were isolated by polymerase chain re-
action (PCR) and sequenced. The deduced amino
acid sequence of full-length CB 1 has CaM-bind-
ing domain and the entire conserved domain of
protein kinase and showed homology with mam-
malian CaM KII.*03These results provide con-
vincing evidence that indicates the existence of
CaMdependent protein kinases in plants and open
indicated that CB 1 represents a rare mRNA, which
is probably one of the reasons why it was not
represented in the pool of various protein kinases
isolated using oligoprobes made to conserved do-
mains of protein kinases. If the mRNA level is
the indication of protein level, one would expect
very low levels of this CaM kinase, which may
have contributed to problems in purifying this
enzyme. Southern analysis of genomic DNA from
Arubidopsis, chicory, and tomato with a CB1
insert (which contains mostly CaM-binding re-
gion) under low stringency conditions showed a
small number of major hybridizing bands, sug-
gesting the presence of similar genes in other
plants. Plants, unlike animal systems, seem to
have both CDPK (discussed later) and Ca2+/CaM-
dependent protein kinases. Having these two dif-
ferent types of protein kinases would explain, to
some extent, how Ca2+ might regulate diverse
physiological processes in plants. Identification
of CaM-dependent protein kinases and their sub-
strates in different plant systems will certainly
continue to be one of the active areas of research
in plants.
Gel overlay assay was used to detect CBP
and their distribution. In Fucus, Brawley and
demonstrated changes in CBP during
development. Oh et al.lS5detected several CBP
in carrot embryo extract. Some of these were
found to change during carrot embryogenesis and
germination.135A CBP of 54 kDa markedly in-
creased during embryo germination. In different

197
 1

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D X P W V V G L S A R E D Q ~ D - A E I V S R Q S F N A R R K
~ ~ ? W I SQ W R Sv5:T Vs n
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CB 1
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$7;
K S L L N

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110 120
CBI
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FIGURE 6. Alignment of deduced amino acid sequence of apple CB1 polypeptide with isoforms
of CaM KII from rat brain.’1~105~1s1~1s2
Identical amino acids between CB1 and any of the CaM KII
subunits are boxed. Dashes indicate single-residue gaps inserted to maximize the alignment of the
apple and mammalian proteins. The arrows indicate the borders of the conserved subdomain XI of
protein kinases.’O The invariant arginine residue found in the domain XI is indicated by an asterisk
(From Watillon, B., Kettmann, R., Boxus, Ph., and Burny, A., Plant Sci., 81, 227, 1992. With per-
mission.)

tissues of Viciafaba and guard cell protoplasts,
CBP were analyzed by gel overlay assay. Sev-
eral CBP that are common to metabolically active
plant parts and that are specific to guard cell
protoplast, stem, and root were detected.’@These
studies indicate that there are several CBP in
plants and some of them are specific to a partic-
ular tissue or cell type.
To better understand the mode of Ca2+/CaM
action, it is essential to identify and characterize
all the CBP in plants. Characterization of various
CBP from animal systems revealed a CaM-bind-
ing domain that contained a basic amphiphilic a
helix.’32 However, there is no amino acid se-
quence conservation in the CaM-binding domain
among different CBP. Hence, it has not been
possible to design oligonucleotideprobes to clone
CBP. Sikela and HahnlS6developed a method to
isolate CBP from a cDNA expression library us-
ing lz5I-labeledCaM. Using this method Watillon
et a1.201isolated a cDNA for CBP that was iden-
tified as a CaM-dependent protein kinase (dis-
cussed earlier). In recent years, this method has
been improved by using 35S-labeledCaM .6,55,206
By screening a corn root tip cDNA expression
library using 35S-labeledCaM as the probe, Reddy
et a1.lw obtained two cDNAs (CBP-1 and CBP-
5) that code for CBP. Comparison of the deduced
amino acid sequence of CBP-1 and CBP-5 clones
showed an overall 50% identity. However, 100%

198
 conservation of the 34 amino acid stretch at their
carboxy-terminal end was observed (Figure 7A).
Hence, this conserved region could be a potential
CaM-binding domain. Analysis of CaM-binding
domains of different CBPs from animal systems
indicates that they all form a basic, amphiphilic
helix. I13.132.133.187.198 In most cases, the CaM-
binding domain contained about 15 to 30 amino
acid residues with a cluster of basic residues ad-
jacent to a hydrophobic region. The highly con-
served 34 amino acid stretch contained the pu-
Downloaded by [University of Illinois at Urbana-Champaign] at 13:00 10 March 2015
tative CaM-binding domain, a basic amphiphilic
CY helix. As shown in the helical wheel plot (Fig-
ure 7B), the putative CaM-binding domain has
a cluster of basic residues (hydrophobic region)
facing hydrophilic amino acids. The fact that the
34 amino acid stretch is highly conserved be-
tween two different CBP clones and forms an
amphiphilic CY helix strongly suggests that it is a
CaM-binding domain in CBP-1 and CBP-5.
However, further studies are needed to confirm
the CaM-binding domain by deletion analysis or

LQXXPWVW 163
cbp5
cbpl E ....... ..... 33

Cbp5 S Q Q T W D W V I 0 P 0 T V T V 8 I L W T S 208
Cbpl ...................... 56

Cbp5 253
Cbpl 100

Cbp5
Cbpl

FIGURE 7A. Alignment of the deduced amino acid sequences of two CaM-binding proteins from corn. Alignment
was made by using the gap program. The underlinedportion shows 100% similarity between the two CaM-binding
proteins and contains the putative CaM-binding region.‘”

0 = Hydrophobic

FIGURE 78. Helical wheel projection of putative CaM-binding of corn CaM-

binding proteins. Amino acid residues from 99-1 16 of CBP-1 and 252-269 of
CBP-5 of 34 amino acid conserved stretch of CBP-1 and CBP-5 were used to
plot the helical wheel.‘”

199
 by competition experiments using synthetic pep-
tides. A number of CBP have been sequenced
from animal systems. A computer search of nu-
cleotide and protein data bases with both nu-
cleotide and deduced amino acid sequences of
CBP-1 and CBP-5 has not revealed any signifi-
cant homology between CBP sequences and
known nucleic acid and protein sequences.
Different CBP isolated from animals indicate
that there is no amino acid sequence conserva-
tion. However, CBP that belong to a particular
class, e.g., different isozymes of CaM-dependent
protein kinases, have the same amino acid se-
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quence in the CaM-binding domain. 192 Hence,
the highly conserved amino acid region in the
putative CaM-binding region of CBP- 1 and CBP-
5 suggests that these two proteins may be related
or perform similar functions.
Northern analysis with both CBP-1 and CBP-
5 clones indicated that the corresponding genes
are expressed in all of the parts tested, although
there was a difference in the extent of expression.
CBP-5 is expressed almost equally in all of the
parts tested, whereas CBP-I mRNA was found
to be differentially expressed in different parts,
with very low levels in the root elongation zone.
The fact that the CBP-1 and CBP-5 are expressed
in all of the parts tested indicates a broader role
for these proteins.
Southern analysis indicates that CBP-1 and
CBP-5 are coded, most likely, by one or two
genes. Depending on the enzyme, two to four
bands were hybridized to CBP clones. However,
the banding pattern observed with CBP-1 is en-
tirely different from CBP-5. Although CBP-1 and
CBP-5 showed 50% identity at the deduced amino
acid level, they do not seem to show cross-hy-
bridization because no common hybridizing bands
were detected between the Southerns of the CBP-
1 and CBP-5 clones.
B. Calcium-Dependent and Calmodulin-
Independent Protein Kinases
CDPK are one of the best characterized and
widely distributed protein kinases in plants. A
CDPK has been purified and characterized from
~ o y b e a n . ~The
” ’ ~native
~ molecular weight of the
CDPK has been determined to be 52.2 kDa by
200
size exclusion chromatography. The CDPK is ac-
tivated by micromolar concentrations of Ca2 and
+
is not dependent on CaM for its activity. The
CDPK showed Ca2 -dependent mobility shift as
+
well as Ca2+ binding.
Recently, Harper et al.73isolated a cDNA
(SK5) from soybean that codes for a CDPK. The
deduced amino acid sequence of soybean CDPK
contains a protein kinase catalytic domain and a
CaM-like region with four Ca2 -binding domains
+
at the carboxy-terminal end. The presence of these
Ca2+ -binding domains explains the direct Ca2 +
activation of CDPK. The kinase domain showed
the highest homology (39%) with the catalytic
domain of the @-subunitof CaM KII. So far, this
new type of protein kinase, where the kinase
domain is fused to a CaM-like region, has been
found only in plants. However, in the case of
calpain, a Ca2+-activated protease, the catalytic
domain is fused to a CaM-like regulatory do-
main.1x9Isolation of a partial cDNA (SK2), which
codes for a protein that shows 70% identity with
CDPK, and the observation that SK5 hybridizes
to multiple fragments on Southern blots indicate
the presence of multiple CDPK isoforms in soy-
bean. Partial cDNAs that code for CDPK have
been isolated from and corn. l 3 Alignment
of the deduced amino acid sequences of the
CDPKs of soybean, carrot, and corn is shown in
Figure 8.
Using a variety of approaches, the presence
of CDPK-like enzymes was shown in a number
of plants, indicating the ubiquitous nature of these
enzymes in plant^.^^^^^,^^,^^^,^^^ CDPKs have been
partially purified from a variety of plant systems.
Biochemical properties and various other aspects
of CDPKs were reviewed recently by Roberts and
Harmon170and hence are not discussed here.
IV. CALCIUM AND GENE EXPRESSION
Studies with animal systems have provided
unequivocal evidence for the role of Ca2+ in the
regulation of gene expression, at both the tran-
scriptional and translational levels. There are sev-
eral instances where Ca2 and/or Ca2 -binding
+ +
proteins have been shown to regulate the expres-
sion of specific genes. 43,91,159,165,183,1XX,204,205
Expression of glucose-regulated genes has been
shown to increase by increasing the cytosolic Ca2+
 Corn
Soy
1
..........
M A A K S S S S S T
TTi;.TiK;I ~JJGHP~PSL
PQR Q N I
~ D H ~
E V E V
A L
45

Carrot ............................................

Corn
SOY
=;;.;;;mi;
I AP LI CT
90

Carrot ............... L vs
Downloaded by [University of Illinois at Urbana-Champaign] at 13:00 10 March 2015

316 V 360
Corn M A
SOY KM
Carrot AN

761 An5

406 450
Corn P A W F
SOY
Carrot N

451 495
Corn V ......................
Soy M KGNGGIG R R T M R K RDALG
Carrot I R A R N R R K
LFSVSLnkN! .....
496 510
Corn ...............
Soy L V D N G S N Q V I E G Y F K
Carrot ...............
FIGURE 8. Alignment of amino acid sequence of Ca2+-dependentand CaM-independent
protein kinase from soybean;73carrot;= and corn.13Arrow indicatesthe beginningof calmodulin-
like domain; calcium-binding sites are underlined.

with CaZ ionophore.'65Several lines of evidence

+ quences upstream of these genes are involved in
exist for the transcriptional regulation of prolactin CaZ -induced response. IS8 Several recent reports
+

gene by CaZ+.9*205 CaZ -induced prolactin gene

+
expression is believed to be mediated by CaM.Zo5
Using gene chimeras consisting of 5' flanking
regions of Ca2+-induced genes linked to the
chloramphenicol acetyltransferase (CAT) re-
porter gene, it was demonstrated that the se-
indicate that Ca2+ regulates the transcription of
target genes by affecting changes in the phos-
phorylation status of specific transcription fac-
tors. In the majority of these cases, Caz+/CaM-
dependent protein kinase I1 was found to phos-
phorylate the transacting factors. Transcription

201
 of a promoter containing the cAMP response ele-
ment (CRE) is enhanced when the cAMP re-
sponse element-binding protein (CREB) is phos-
phorylated by Ca2 /CaM-dependent protein
+ Ca2+ concentrations, whereas an increase in Mg2
kinase II.43,183Phosphorylation of CEBP ( C U T /
enhancer binding protein) and C/EBPP (leucine
zipper) has been shown to confer Ca2 -regulated
+
transcriptional stimulation of promoters contain-
ing the binding sites for these transcription fac-
tor~.~~,~@'
In addition, some of the Ca2+-mediated
changes in gene expression have been attributed
to the phosphorylation of transcription factors by
protein kinase C.19,62The binding of heat shock
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factor (HSF) to the cis-regulatory elements in
inducing the heat shock genes also requires
Ca2+.125,151
In addition to transcriptional control, Ca2+
also plays a role in translational control. Eu-
karyotic elongation factor-2 (eEF-2), one of the
elongation factors of protein synthesis, serves as
a substrate for Ca2+ and CaM-dependent protein
kinase I11 (Ca2+/CaMKIII also is called eEF-2
Phosphorylation of eEF-2 by the
kinase). 131~174~175
Ca2+/CaMKIII makes it inactive, which results
in the drastic inhibition of the elongation rate of
translation.174 There also is evidence implicating
a role for Ca2+ in the initiation of protein syn-
thesis.25,26,32,52
Ca2+ depletion was found to de-
crease the rate of initiation of protein synthesis.32
Inhibition of protein synthesis in GH3 pituitary
cells by Ca2+ depletion is correlated with de-
phosphorylation of a 26-kDa ribosome-associ-
ated protein.52
Based on indirect and circumstantial evi-
dence, we proposed that Ca2+ either directly or
indirectly regulates the expression of specific
genes in plants. 145 Evidence obtained in recent
years indicates regulation of gene expression in
plants by Ca2 . There are a few examples of plant
+ needed to determine which of the steps (tran-
genes whose expression is specifically altered by
experimental manipulation of cellular Ca2 lev-
+ stability) is controlled by Ca2 .
els. Lam et al.lol showed the induction of chlo-
rophyll a- and b-binding (CAB) protein mRNA
levels in the dark by increasing the intracellular
Ca2+ level with ionomycin. However, the in-
duction is only 10% as compared to that induced
by light. Using CaM antagonists, they implied
the possible involvement of CaM in phyto-
chrome-regulated CAB protein gene expression.
However, it should be noted that the CaM an-
tagonists are known to inhibit the activity of pro-
202
teins other than CaM. 145~170The transcripts corre-
sponding to some of the touch genes were
enhanced in response to an increase in external
+
had no effect.22 Heat induction of touch gene
expression was blocked by Ca2+ depletion and
restored by Ca2+ replenishment. However, ex-
ternal Ca2+ is not required for the induction of
heat shock proteins such as hsp-75 and hsp-
70.22*101Based on these results, it has been sug-
gested that signals that induced touch genes el-
evate cytosolic Ca2+ levels, which in turn reg-
ulate the expression of touch genes.22In addition,
these results suggest that Ca2+ could regulate
expression of the genes that encode its receptors
because some of the touch genes code for CaM-
like proteins. Knight et al. ,95 using transgenic
tobacco seedlings that express aequorin, showed
that touch signal induced rapid and transient in-
creases in cytosolic Ca2+, although heat shock
did not show any changes in cytosolic Ca2+. In
animal cells, heat shock elevates cytosolic Ca2
+
by releasing Ca2+ from internal ~ t o r e s .Raz~~,~~
and F l ~ h r showed
' ~ ~ that the chitinase induction
by ethylene is mediated by Ca2+ . Ca2+ depletion
with the Ca2+ chelator EGTA blocked the eth-
ylene-dependent induction of chitinase, whereas
the ethylene-independent induction of chitinase
was unaffected by Ca2+depletion. Furthermore,
increasing cytosolic Ca2 by ionophore (iono-
+
mycin) or microsomal Ca2 -ATPase inhibitor
+
(thapsigargin) induced chitinase accumulation in
the absence of ethylene. These results strongly
suggest that Ca2+regulates the expression of the
chitinase gene and mediates the action of ethyl-
ene. Whether CaM is involved in the Ca2+ in-
duction of touch and the chitinase genes is not
known at the present time. Further research is
scription, mRNA stability, translation, or protein
+
We explored the possibility of regulation of
CaM gene expression by Ca2+in transgenic plants
carrying the rice CaM-2 promoter fused to the
CAT reporter gene. Depletion of Ca2+ by EGTA
and ionomycin resulted in the decreased expres-
sion of CAT activity. Plants carrying the CaMV
35s promoter with a reporter gene CAT was used
as the control. Ca2+ depletion did not affect the
CAT expression driven by the CaMV 35s pro-
moter. Furthermore, verapamil, a Ca2+ channel
 blocker, also decreased the expression of the re-
porter gene driven by the CaM-2 promoter.162
These results suggest a role for Ca2+in regulating
the transcription of the rice CaM-2 gene. In rice,
there are about four genes that code for CaM.
We do not know whether other genes are regu-
lated in a similar manner. In Arubidopsis, CaM
is coded by multiple genes. In Arabidopsis cul-
tures, the expression of one of the CaM genes
(TCH 1) is unaffected by increased external Ca2+
and is induced by heat shock. It is likely that
promoters of different CaM genes in a system
have distinct regulatory elements that respond
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differentially to various signals.
Although there are only a few examples of
Ca2+-regulated genes in plants, it is likely that
this list will grow because of the advances in
molecular biology. Having identified some Ca2 -+
responsive genes, it is now possible to identify
the cis-elements involved in Ca2+ induction as
well as the mechanisms by which Ca2+regulates
the expression of specific genes.
V. CONCLUSION AND FUTURE
PROSPECTS
From the literature presented here, it is evi-
dent that Ca2+plays a central role as a messenger
and regulates many aspects of cellular signaling.
It is now clear that many physical and hormonal
signals (touch, wind, gravity, light, cold, auxin,
GA, ABA, salt, and fungal elicitors) induce rapid
changes in cytosolic Ca2+levels and these changes
precede the physiological responses. Although
the signals induce an increase in cytosolic Ca2+
in a majority of cases, there are instances where
the cytosolic Ca2+ level is decreased by sig-
n a l ~ .Furthermore,
~ ~ , ~ ~ ~ the same signal may have
the opposite effect on cytosolic Ca2+,depending
on the tissue or cell type.60,67,119,199
The role of
intra- and extracellular Ca2+ pools and Ca2+
channels in elevating cytosolic Ca2+in response
to signals will be one of the key questions that
need to be addressed in future. Having estab-
lished Ca2+ as a messenger in signal transduc-
tion, there are many more questions that need to
be addressed. Most important of all is to under-
stand the precise mechanisms by which an in-
creased cytosolic Ca2 level evoke the response.
+
Two Ca2+ -modulated proteins, CaM and CDPK,
have been well characterized and there are in-
dications of more Ca2+ -modulated proteins, in-
cluding CaM-like proteins, in plants. The ubiq-
uitous presence of CDPKs in plants and their
heterogeneity indicate a broader, important role
in signal transduction as well as complexity of
these enzymes in regulating cellular processes.
Recent studies indicate the presence of CaM-de-
pendent protein kinases in plants, although it is
not known how widely these kinases are present
in plants. Identification and characterization of
the target proteins that interact with Ca2+ alone
or the Ca2+/CaM complex as well as the sub-
strates for CDPKs and CaM kinases are crucial
to elucidate the signal transduction pathways. So
far, very few CaM target proteins are known in
plants as compared to animals. The new approach
of isolating cDNA clones for CBP by screening
expression libraries with radiolabeled CaM will
expedite the progress and increase our knowledge
about CaM target proteins in plants. Because Ca2+
is known to be involved in regulating diverse
physiological processes, it is likely that different
tissues or cell types may have different types of
Ca2+ target proteins as well as substrates for
CDPKs and CaM-regulation protein kinases.
Identification and characterization of CDPK type
and CaM-dependent protein kinases and their
substrates will certainly contribute to further un-
derstanding of Ca2+action in plants in regulating
numerous functions during plant growth and de-
velopment.
Although the Ca2+ -mediated signaling path-
ways in plants appear to be similar to those de-
scribed in animals, there are several features that
are unique to plants and suggest that there are
major differences in the mode of Ca2 -signalling
+
pathways between plants and animals. In plants,
vacuolar Ca2+ stores appear to play an important
role in increasing the cytosolic Ca2+ concentra-
tion in response to signals, whereas in animal
systems, ER plays a major role in contributing
to increases in cytosolic Ca2 levels. The expres-
+
sion of CaM and CaM-like proteins is highly
responsive to various physical and chemical sig-
n a l ~ .In~ addition,
~ . ~ ~ Ca2+ seems to regulate the
expression of the genes that code for its recep-
tors.22,162
Plants have a unique type of protein
kinase in which a catalytic domain is fused to a
CaM-like domain. Hence, these kinases
13733373,170
are regulated by Ca2+ alone in a CaM-indepen-
203
 dent manner. So far, this group of protein kinases
has been reported only in plants and Parume-
cium, whereas in animals, there are several types
of Ca2+/CaM-dependentprotein kinases.
ACKNOWLEDGMENTS
The support of National Science Foundation
(Grant No. DCB-9104586), National Aeronau-
tics and Space Administration (NAG-10-0061 S/
l ) , and the Agricultural Experiment Station
(Project No. 0321) to B.W.P. and Agricultural
Downloaded by [University of Illinois at Urbana-Champaign] at 13:00 10 March 2015
Experiment Station (Project No. 702) and the
Plant Biotechnology Laboratory to A.S.N.R. is
gratefully acknowledged. The authors are in-
debted to all researchers who worked with us on
this project. Our special thanks to Meg Gallagher
and Nadine Kuehl for their assistance in typing
the manuscript and Lee Turner for his help in the
literature search.
REFERENCES
1. Alexandre, J., Lassalles, J. P., and Kado, R. T.,
Opening of Ca2+ channels in isolated red beet root
vacuole membrane by inositol 1,4,5-triphosphate,
Nature, 343, 567, 1990.
2. Anderson, J. M., Charbonneau, H., Jones, H. P.,
McCann, R. O., and Cormier, M. J., Character-
ization of the plant nicotinamide adenine dinucleotide
kinase activator protein and its identification as cal-
modulin, Biochemistry, 19, 3113, 1980.
3. Anderson, J. M. and Cormier, M. J., Calcium-
dependent regulator of NAD kinase in higher plants,
Biochem. Biophys. Res. Commun., 84, 595, 1978.
4. Andrejauskas, E., Hertel, R., and Marme, D.,
Specific binding of the calcium antagonist [)HI ve-
rapamil to membrane fractions from plants, J. Biol.
Chem., 260, 5411, 1985.
5. Asaoka, Y., Nakamura, S., Yoshida, K., and
Nishizuka, Y., Protein kinase C, calcium and phos-
pholipid degradation, Trends Biochem. Sci., 17,414,
1992.
6. Asselin, J., Phaneuf, S., Watterson, D. M., and
Haiech, J., Metabolically 35S-labeledrecombinant
calmodulin as a ligand for the detection of calmo-
dulin-binding proteins, Anal. Biochem., 178, 141,
1989.
7. Bachs, 0. and Carafoli, E., Calmodulin and cal-
modulin-binding proteins in liver cell nuclei, J. Biol.
Chem., 262, 10786, 1987.
204
8. Bachs, O., Lanini, L., Serratosa, J., Coil, M. J.,
Bastos, R., Aligue, R., Rius, E., and Carafoli, E.,
Calmodulin-binding proteins in the nuclei of quies-
cent and proliferatively activated rat liver cells, J.
Biol. Chem., 265, 18595, 1990.
9. Bandyopadhyay, S. K. and Bancroft, C., Calcium
induction of the mRNAs for prolactin and clfos is
independent of protein kinase C activity, J. Biol.
Chem., 264, 14216, 1989.
10. Barnett, M. J. and Long, S. R., Nucleotide se-
quence of an alfalfa calmodulin cDNA, Nucleic Acids
Res., 18, 3395, 1990.
11. Bennett, M. K. and Kennedy, M. B., Deduced
primary structure of the P-subunit of brain type I1
Ca*+/calmodulin-dependentprotein kinase deter-
mined by molecular cloning, Proc. Natl. Acad. Sci.
U.S.A., 84, 1794, 1987.
12. Berridge, M. J. and Irvine, R. F., Inositol phos-
phates and cell signaling, Nature, 341, 197, 1989.
13. Bhatia, A., Patil, S., and Poovaiah, B. W., un-
published results.
14. Birch, B. D., Eng, D. L., and Kocsis, J. D., In-
tranuclear CaZ transients during neurite regeneration
+
of an adult mammalian neuron, Proc. Natl. Acud.
Sci. U.S.A., 89, 7978, 1992.
15. Blatt, M. R., Thiel, G., and Trentham, D. R.,
Reversible inactivation of K + channels of Vicia sto-
matal guard cells following the photolysis of caged
inositol 1,4,5-trisphosphate, Nature, 346,766, 1990.
16. Blowers, D. P., Hetherington, A., and Trewavas,
A. J., Isolation of plasma membrane bound calcium/
calmodulin dependent protein kinase from pea and
modification of catalytic activity by autophosphory-
lation, Biochem. Biophys. Res. Commun., 143, 691,
1985.
17. Blowers, D. P. and Trewavas, A. J., Rapid cycling
of autophosphorylationof Ca2 -calmodulin regulated
+
plasma membrane located protein kinase from pea,
Plant Physiol., 90, 1279, 1989.
18. Blum, W., Hinsch, K.-D., Schultz, G., and Weiler,
E. W., Identification of GTP-binding proteins in the
plasma membrane of higher plants, Biochem. Bio-
phys. Res. Commun., 156, 954, 1988.
19. Bohmann, D., Bos, T. J., Admon, A., Nishimura,
T., Vogt, P. K., and Tjian, R., Human proto-on-
cogene c-jun encodes a DNA binding protein with
structural and functional properties of transcription
factor AP-1, Science, 238, 1386, 1987.
20. Boss, W. F., Second messengers in plant growth and
development, in Plant Biology, Vol. 6, Boss, W. F.
and Morre, D. J., Eds., Alan R. Liss, New York,
1989, 29.
21. Braam, J. and Davis, R. W., Rain-, wind-, and
touch-induced expression of calmodulin and calmo-
dulin-related genes in Arabidopsis, Cell, 60, 357,
1990.
22. Braam, J., Regulated expression of the calmodulin-
related TCH genes in cultured Arabidopsis cells: in-
duction by calcium and heat shock, Proc. Natl. Acud.
Sci. U.S.A., 89, 3213, 1992.
 23. Brawley, S. H. and Roberts, D. M., Calmodulin-
binding proteins are developmentally regulated in ga-
metes and embryos of fucoid algae, Dev. Biol., 131,
313, 1989.
24. Brosnan, J. M. and Sander, D., Inositol trisphos-
phate mediated Ca2+ release in beet microsomes is
inhibited by heparin, FEBS Lett., 260, 70, 1990.
25. Brostrom, C. O., Chin, K. V., Wong, W. L., Cade,
C., and Brostrom, M. A., inhibition of translational
initiation in eukaryotic cells by calcium ionophore,
J. Biol. Chem., 264, 1644, 1989.
26. Brostrom, M. A., Lin, X., Cade, C., Gmitter, D.,
and Brostrom, C. O., Loss of a calcium requirement
for protein synthesis in pituitary cells following ther-
mal or chemical stress, J. Biol. Chem., 264, 1638,
Downloaded by [University of Illinois at Urbana-Champaign] at 13:00 10 March 2015
1989.
27. Brownlee, C. and Wood, J. W., A gradient of cy-
toplasmic free calcium in growing rhizoid cells of
Fucus serratus, Nature, 320, 624, 1986.
28. Brownlee, C. and Pulsford, A. L., Visualization
of the cytoplasmic Caz+ gradient in Fucus serratus
rhizoids: correlation with cell ultrastructure and po-
larity, J. Cell Sci., 91, 249, 1988.
29. Bush, D. S. and Jones, R. L., Cytoplasmic calcium
and a-amylase secretion from barley aleurone pro-
toplasts, E. J. Cell Biol., 46, 466, 1988.
30. Bush, D. S. and Jones, R. L., Measuring intracel-
lular Ca2+ levels in plant cells using the fluorescent
probes, indo-1 and fura-2, Plant Physiol., 93, 841,
1990.
31. Calderwood, S. K., Stevenson, M. A., and Hahn,
G. M., Effects of heat on cell calcium and inositol
lipid metabolism, Radiat. Res., 113, 414, 1988.
32. Chin, K.-V., Cade, C., Brostrom, C. O., Galuska,
E. M., and Brostrom, M. A., Calcium-dependent
regulation of protein synthesis at translational initi-
ation in eukaryotic cells, J. Biol. Chem., 262, 16509,
1987.
33. Choi, J. H. and Suen, K.-L., Isolation and sequence
analysis of a cDNA for a carrot calcium-dependent
protein kinase: homology to calciudcalmodulin-de-
pendent protein kinases and calmodulin, Plant Mol.
Biol., 17, 581, 1991.
34. Choi, Y. J., Poovaiah, B. W., and An, G., un-
published results.
35. Chua, N. H., Carlenor, E., and Fromm, H., un-
published results from Genbank.
36. Clark, G. B., Dauwalder, M., and ROUX,S. J.,
Purification and immunolocalization of an annexin-
like protein in pea seedlings, Planta, 187, 1, 1992.
37. Clarkson, D. T., Brownlee, C., and Ayling, S. M.,
Cytoplasmic calcium measurements in intact higher
plant cells: results from fluorescence ratio imaging
of fura-2, J. Cell Sci., 91, 71, 1988.
38. Clowell, G., Paul, E. M., Thomas, S. R., and
Erion, J. L., unpublished results from Genbank.
39. Cohen, P., The structure and regulation of protein
phosphatases, in The Biology and Medicine of Signal
Transduction, Nishizuka, Y., Ed., Raven Press, New
York, 1990, 230.
40. Cohen, P., Signal integration at the level of protein
kinases, protein phosphatases and their substrates,
Trends Biochem. Sci., 17, 408, 1992.
41. Colbran, R. J. and Soderling, T. R., Calciudcal-
modulin-dependent protein kinase 11, Curr. Top. Cell.
Regul., 31, 181, 1990.
42. Cosgrove, D. J. and Hedrich, R., Stretch-activated
chloride, potassium, and calcium channels coexisting
in plasma membranes of guard cells of Viciafaba L.,
Planta, 186, 143, 1991.
43. Dash, P. K., Karl, K. A., Colicos, M. A., Prywes,
R., and Kandel, E. R., CAMP response element-
binding protein is activated by Caz+/calmodulin-as
well as CAMP-dependent protein kinase, Proc. Natl.
Acad. Sci. U.S.A., 88, 5061, 1991.
44. Davis, T. N., Urdea, M. S., Masiarz, F. R., and
Thorner, J., Isolation of the yeast calmodulin gene:
calmodulin is an essential protein, Cell, 47, 423,
1986.
45. Davis, T. N. and Thorner, J., Vertebrate and yeast
calmodulin, despite significant sequence divergence,
are functionally interchangeable, Proc. Natl. Acad.
Sci. U.S.A., 86, 7909, 1989.
46. Ding, J. P. and Pickard, B. G., Mechanosensory
calcium-selective cation channels in epidermal cells,
Plant J . , in press.
47. Drummond, I. A. S., McClure, S. A., Poenie, M.,
Tsien, R. Y.,and Steinhardt, R. A., Large changes
in intracellular pH and calcium observed during heat
shock are not responsible for the induction of heat
shock proteins in Drosophila melanogaster, Mol. Cell.
Biol., 6, 1767, 1986.
48. Echevarria, C., Vidal, J., Le Marechal, P.,
Brulfert, J., and Ranjeva, R., The phosphorylation
of Sorghum leaf phosphoenolpyruvate carboxylase is
a Ca+ +-calmodulin dependent process, Biochem.
Biophys. Res. Commun., 155, 835, 1988.
49. Einspahr, K. J. and Thompson, G. A., Jr., Trans-
membrane signaling via phosphatidylinositol 4,5 ,-
bisphosphate hydrolysis in plants, Plant Physiol., 93,
361, 1990.
50, Evans, D. E., Briars, S. A., and Williams, L. E.,
Active calcium transport by plant cell membranes, J .
Exp. Bot., 42, 236, 1991.
51. Fairley-Grenot, K. and Assmann, S. M., Evidence
for G-protein regulation of inward K + channel current
in guard cells of fava bean, Plant Cell, 3,1037,1991.
52. Fawell, E. H., Boyer, I. J., Brostrom, M. A., and
Brostrom, C. O., A novel calcium-dependent phos-
phorylation of a ribosome-associated protein, J. Biol.
Chem., 264, 1650, 1989.
53. Feldman, L. J., Root gravitropism, Physiol. Plant.,
65, 341, 1985.
54. Fischer, R., Koller, M., Flura, M., Mathews, S.,
Strehler-Page, M.-A., Krebs, J., Penniston, J. T.,
Carafoli, E., and Strehler, E. E., Multiple diver-
gent mRNA as code for a single human calmodulin,
J. Biol. Chem., 263, 17055, 1988.
55. Fromm, H. and Chua, N.-H., Cloning of plant
cDNAs encoding calmodulin-binding proteins using
 35S-labeledrecombinant calmodulin as a probe, Plant
Mol. Biol. Rep., 12, 199, 1992.
56. Fromm, H., personal communication.
57. Fujusawa, H., Calmodulin-dependent protein kinase
11, BioEssays, 12, 27, 1990.
58. Gawienowski, M. C., Szymanski, D., Perera, I. Y.,
and Zielinski, R. E., unpublished results from Gen-
bank.
59. Gehring, C. A.,.Williams, D. A., Cody, S. H.,
and Parish, R. W., Phototropism and geotropism in
maize coleoptiles are spatially correlated with in-
creases in cytosolic free calcium, Nature, 345, 528,
1990.
60.Gehring, C. A., Irving, H. R., and Parish, R. W.,
Effects of auxin and abscisic acid on cytosolic cal-
Downloaded by [University of Illinois at Urbana-Champaign] at 13:00 10 March 2015
cium pH in plant cells, Proc. Natl. Acad. Sci. U.S.A.,
87, 9645, 1990.
61. Geiser, J. R., van Tuinen, D., Brickerhoff, S. E.,
Neff, M. M., and Davis, T. N., Can calmodulin
function without binding calcium?, Cell, 65, 949,
1991.
62. Ghosh, S. A. and Baltimore, D. B., Activation in
vitro of NF-KB by phosphorylation of its inhibitor
IKB, Nature, 344, 678, 1990.
63. Gilroy, S., Blowers, D. P., and Trewavas, A. J.,
Calcium: a regulation system emerges in plant cells,
Development, 100, 181, 1987.
64. Gilroy, S., Hughes, W. A., and Trewavas, A. J.,
A comparison between quin-2 and aequorin as in-
dicators of cytoplasmic calcium levels in higher plant
cell protoplasts, Plant Physiol., 90, 482, 1989.
65, Gilroy, S., Read, N. D., and Trewavas, A. J.,
Elevation of cytoplasmic Caz+ by caged calcium or
caged inositol trisphosphate initiates stomata1 clo-
sure, Nature, 346, 769, 1990.
66. Gilroy, S., Fricker, M. D., Read, N. D., and
Trewavas, A. J., Role of calcium in signal trans-
duction of commelina guard cells, Plant Cell, 3, 333,
1991.
67. Gilroy, S. and Jones, R. L., Gibberellic acid and
abscisic acid coordinately regulate cytoplasmic cal-
cium and secretory activity in barley aleurone pro-
toplasts, Proc. Natl. Acad. Sci. U.S.A., 89, 3591,
1992.
68. Graziana, A., Fosset, M., Ramjeva, R.,
Hetherington, A. M., and Lazdunski, M., Ca2+
channel inhibitors that bind to plant cell membranes
block Caz+ entry into protoplasts, Biochemistry, 27,
764, 1988.
69. Gregori, L., Marriott, D., Putkey, J. A., Means,
A. R., and Chau, V., Bacterially synthesized ver-
tebrate calmodulin is a specific substrate for ubiqui-
tination, J. Biol. Chem., 262, 2562, 1987.
70. Hanks, S. K., Quinn, A. M., and Hunter, T., The
protein kinase family: conserved features and de-
duced phylogeny of the catalytic domains, Science,
241, 42, 1988.
71. Harmon, A. C., Putnam-Evans, C., and Cormier,
M. J., A calcium-dependent but calmodulin-inde-
206
pendent protein kinase from soybean, Plant Physiol.,
83, 830, 1987.
72. Harmon, A. C. and McCurdy, W., Calcium-de-
pendent protein kinase and its possible role in the
regulation of the cytoskeleton, in Current Topics in
Plant Biochemistry and Physiology, Vol. 9, Plant
Protein Phosphorylation, Protein Kinases, Calcium
and Calmodulin, Randall, D. D. and Belvins, D. G.,
Eds., University of Missouri, Columbia, 1990, 119.
73. Harper, J. F., Sussman, M. R., Schaller, G. E.,
Putnam-Evans, C., Charbonneau, H., and
Harmon, A. C., A calcium-dependent protein kinase
with a regulatory domain similar to calmodulin, Sci-
ence, 252, 951, 1991.
74. Harvey, H. J., Venis, M. A., and Trewavas, A. J.,
Partial purification of a protein from maize coleoptile
membranes binding the calcium channel antagonist
verapamil, Biochem. J., 257, 95, 1989.
75. Hasunuma, K., Furaukawa, K., Funadera, K.,
Kubota, M., and Watanabe, M., Partial charac-
terization and light-induced regulation of GTP-bind-
ing proteins in Lemna paucicostata, Photochem.
Photobiol., 46, 531, 1987.
76. Hasunuma, K., Furaukawa, K., Tomita, K.,
Mukai, C., and Nakamura, T., GTP-binding pro-
teins in etiolated epicotyls of Pisum sativum (Alaska)
seedlings, Biochem. Biophys. Res. Comrnun., 148,
133, 1987.
77. Heizmann, C. W. and Hunziker, W., Intracellular
calcium-binding proteins: more sites than insights,
Trends Biochem. Sci., 16, 98, 1991.
78. Hepler, P. K. and Wayne, R. O., Calcium and plant
development, Annu. Rev. Plant Physiol., 36, 397,
1985.
79. Hepler, P. K. and Callaham, D. A., Free calcium
increases during anaphase in stamen hair cells of Tra-
descantia, J. Cell Biol., 105, 2137, 1987.
80 Hepler, P. K., Does calcium regulate events through
amplitude modulation?, in Current Topics in Plant
Biochemistry and Physiology, Vol. 9, Plant Protein
Phosphorylation, Protein Kinases, Calcium and Cal-
modulin, Randall, D. D. and Belvins, D. G., Eds.,
University of Missouri, Columbia, 1990, 1.
81. Herrera-Estrella, L. and Simpson, J., Foreign gene
expression in plants, in Plant Molecular Biology -
A Practical Approach, Shaw, C. H., Ed., IRL Press,
Oxford, 1988, 131.
82. Hess, P., Calcium channels in vertebrate cells, Annu.
Rev. Neurosci., 13, 337, 1990.
83. Hiatt, A., Antibodies produced in plants, Nature,
344, 469, 1990.
84. Hsieh, W.-L., Pierce, W. S., and Sze, H., Calcium-
pumping ATPases in vesicles from carrot cells, Plant
Physiol., 97, 1535, 1991.
85. Irvine, R., Messenger gets the green light, Nature,
346, 700, 1990.
86. Irving, H. R., Gehring, C. A., and Parish, R. W.,
Changes in cytosolic pH and calcium pH and calcium
 of guard cell precede stomatal movements, Proc. Natl.
Acad. Sci. U.S.A., 89, 1790, 1992.
87. Jaffe, L. F., Calcium explosions as triggers of de-
velopment, Ann. N.Y.Acad. Sci., 339, 86, 1980.
88. Janis, R. A. and Triggle, D. J., Drugs acting on
calcium channels, in Calcium Channels: Their Prop-
erties, Functions, Regulation, and Clinical Rele-
vance, Hunvitz, L., Patridge, L. D., and Leach, J. K.,
Eds., CRC Press, Boca Raton, FL, 1991, 195.
89. Jena, P. K., Reddy, A. S. N., and Poovaiah, B. W.,
Molecular cloning and sequencing of a cDNA for
plant calmodulin: signal-induced changes in the
expression of calmodulin, Proc. Natl. Acad. Sci.
U.S.A., 86, 3644, 1989.
90. Johannes, E., Brosnan, J. M., and Sander, D.,
Downloaded by [University of Illinois at Urbana-Champaign] at 13:00 10 March 2015
Calcium channels and signal transduction in plant
cells, BioEssays, 13, 331, 1991.
91. Kapiloff, M. S., Mathis, J. M., Nelson, C. A.,
Lin, C. R., and Rosenfeld, M. G., Calciudcal-
modulin-dependent protein kinase mediates a path-
way for transcriptional regulation, Proc. Natl. Acad.
Sci. U.S.A., 88, 3710, 1991.
92. Keith, C. H., Ratan, R., Maxfield, F. R., Bajer,
A., and Shelanski, L., Local cytoplasmic calcium
gradients in living mitotic cells, Nature, 316, 848,
1985.
93. Klee, C. B., Concerted regulation of protein phos-
phorylation and dephosphorylation by calmodulin,
Neurochem. Res., 16, 1059, 1991.
94. Klee, C. B., Crouch, T. H., and Richman, P. G.,
Calmodulin, Annu. Rev. Biochem., 49, 489, 1980.
95. Knight, M. R., Campbell, A. K., Smith, S. M.,
and Trewavas, A. J., Transgenic plant aequorin re-
ports the effects of touch and cold-shock and elicitors
on cytoplasmic calcium, Nature, 352, 524, 1991.
96. Knight, M. R., Smith, S. M., and Trewavas, A. J.,
Wind-induced plant motion immediately increases
cytosolic calcium, Proc. Natl. Acad. Sci. U.S.A.,
89, 4967, 1992.
97. Koller, M., Schnyder, B., and Strehler, E. E.,
Structural organization of the human CAM111 cal-
modulin gene, Biochim. Biophys. Acta, 1087, 180,
1990.
98. Krause, K.-H., Chou, M., Thomas, M. A.,
Sjolund, R. D., and Campbell, K. P., Plant cells
contain calsequestrin, J . Biol. Chem., 264, 4269,
1989.
99. Kreimer, G., Melkonian, M., Hokum, J. A. M.,
and Latzko, E., Characterization of Ca2+ fluxes
across the envelope of intact spinach chloroplasts,
Planta, 166, 515, 1985.
100. Kreimer, G., Melkonian, M., and Latzko, E., An
electrogenic uniport mediates light-dependent Ca2 +
influx into intact spinach chloroplasts, FEBS Lett.,
180, 253, 1985.
101. Lam, E., Benedyk, M., and Chua, N.-H., Char-
acterization of phytochrome-regulated gene expres-
sion in a photoautotrophic cell suspension: possible
role for calmodulin, Mol. Cell. Biol., 9, 4819, 1989.
102. Lee, Y. and Assmann, S. M., Diacylglycerols in-
duce both ion pumping in patch-clamped guard-cell
protoplasts and opening of intact stomata, Proc. Natl.
Acad. Sci. U.S.A., 88, 2127, 1991.
103. Lehle, L., Phosphatidyl inositol metabolism and its
role in signal transduction in growing plants, Plant
Mol. Biol., 15, 647, 1990.
104. Li, H., Dauwalder, M., and ROUX,S. J., Partial
purification and characterization of a Caz+-dependent
protein kinase from pea nuclei, Plant Physiol., 96,
720, 1991.
105. Lin, C. R., Kapiloff, M. S., Durgerian, S.,
Tatemoto, K., Russo, A. F., Hanson, P., Schulman,
H., and Rosenfeld, M. G., Molecular cloning of a
brain-specific calciudcalmodulin-dependent protein
kinase, Proc. Natl. Acad. Sci. U.S.A., 84, 5962,
1987.
106. Lin, Z., Pan, N., and Chen, Z., Cloning and se-
quencing of calmodulin cDNA from rice (Oryza sa-
tiva), Acta Bot. Sin.,35, 363, 1991.
107. Ling, V. and Zielinski, R. E., Cloning of cDNA
sequences encoding the calcium-binding protein, cal-
modulin, from barley (Hordeum vulgare L.), Plant
Physiol., 90, 714, 1989.
108. Ling, V., Perera, I., and Zielinski, R. E., Primary
structures of Arabidopsis calmodulin isofoms de-
duced from the sequences of cDNA clones, Plant
Physiol., 96, 1196, 1991.
109. Ling, V. and Assmann, S., Cellular distribution of
calmodulin and calmodulin-binding proteins in Vicia
faba L., Plant Physiol., 100, 970, 1992.
110. Ling, V. and Zielinski, R. E., Isolation of an Ar-
abidopsis cDNA sequence encoding a 22-kilodalton
calcium-binding protein (CaBP-22) related to cal-
modulin, Plant Mol. Biol., in press.
111. Lu, Y. T. and Harrington, M., Isolation and char-
acterization of cDNA clones encoding heat shock-
induced d calmodulin-binding proteins, Plant Phys-
iol. (Suppl.), 96, 397, 1991.
112. Lukas, T. J., Iverson, D. B., Schleicher, M., and
Watterson, D. M., Structural characterization of a
higher plant calmodulin: Spinacea oleracea, Plant
Physiol., 75, 788, 1984.
113. Lukas, T. J., Burgess, W. H., Prendergast, F. G.,
Lau, W., and Watterson, D. M., Calmodulin bind-
ing domains: characterization of a phosphorylation
and calmodulin binding site from myosin light chain
kinase, Biochemistry, 25, 1458, 1986.
114. Lynch, J., Polito, V. W., and Luchli, A., Salinity
stress increases cytoplasmic Ca activity in maize root
protoplasts, Plant Physiol., 90, 1271, 1989.
115. Ma, H., Yanofsky, M. F., and Meyerowitz, E. M.,
Molecular cloning and characterization of GPA1, a
G protein subunit gene from Arabidopsis thaliana,
Proc. Natl. Acad. Sci. U.S.A., 87, 3821, 1990.
116. Mansfield, T. A., Hetherington, A. M., and
Atkinson, C. J., Some current aspects of stomatal
physiology, Annu. Rev. Plant Physiol. Plant Mol.
Biol., 41, 55, 1990.
207
 117. Mazzanti, M., De Felice, L. J., Cohen, J., and
Malter, H., Ion channels in the nuclear envelope,
Nature, 343, 764, 1990.
118. McAinsh, M. R., Brownlee, C., and Hetherington,
A. M., Abscisic acid-induced elevation of guard cell
cytosolic Caz precedes stomatal closure, Nature,
+ pha-helical periodicity, Science, 236, 1454, 1987.
343, 186, 1990.
119. McAinsh, M. R., Brownlee, C., and Hetherington,
A. M., Visualizing changes in cytosolic-free Ca2+
during the response of stomatal guard cells to abscisic
acid, Plant Cell, 4, 1113, 1992.
120. Meader, W. E., Means, A. R., and Quiocho, F. A.,
Target enzyme recognition by calmodulin: 2,4 A
structure of a calmodulin-peptide complex, Science,
257, 1251, 1992.
Downloaded by [University of Illinois at Urbana-Champaign] at 13:00 10 March 2015
121. Miller, D. D., Callahan, D. A., Gross, D. J., and
Hepler, P. D., Free Ca2+gradient in growing pollen
tubes of Lilium, J. Cell Sci., 101, 7, 1992.
122. Miller, A. J. and Sanders, D., Depletion of cyto-
solic free calcium induced by photosynthesis, Nature,
346, 397, 1987.
123. Moncrief, N. D.,Krestinger, R. H., and Goodman,
M., Evolution of EF-hand calcium-modulated pro-
teins. I. Relationships based on amino acid se-
quences, J. Mol. Evol., 30, 522, 1990.
124. Morse, M. J., Satter, R. L., Crain, R. C., and
Cote, G. G., Signal transduction and phosphatidyl-
inositol turnover in plants, Physiol. Plant., 76, 118,
1989.
125. Mosser, D. D., Kotzbauer, P. T., Sarge, K. D.,
and Morimoto, R. I., In vitro activation of heat
shock transcription factor DNA-binding by calcium
and biochemical conditions that affect protein con-
formation, Proc. Natl. Acad. Sci. U.S.A., 87, 3748,
1990.
126. Muto, S. and Miyachi, S., Properties of a protein
activator of NAD kinase from plants, Plant Physiol.,
59, 5 5 , 1977.
127. Neer, E. J. and Clapham, D. E., Roles of G protein
subunits in transmembrane signaling, Nature, 333,
129, 1988.
128. Nicotera, P., McConkey, D. J., Jones, D. P., and
Orrenius, S., ATP stimulates Caz+ uptake and in-
creases the free Caz+ concentration in isolated rate
liver nuclei, Proc. Natl. Acad. Sci. U.S.A., 86,453,
1989.
129. Nishizuka, Y., The molecular heterogeneity of a pro-
tein kinase C and its implication for cellular regu-
lation, Nature, 334, 661, 1988.
130. Nobiling, R. and Reiss, H.-D., Quantitative analysis
of calcium gradients and activity in growing pollen
tubes of Lilium longiflorum, Protoplasma, 139, 20,
1987.
131. Nygard, 0. N. A., Carlberg, U., Nilsson, L., and
Amons, R., Phosphorylation regulates the activity
of the eEF-2-specific Caz+- and calmodulin-depen-
dent protein kinase 111, J. Biol. Chem., 266, 16425,
1991.
132. O’Neil, K. T. and De Grado, W. F., How calmo-
dulin binds its targets: sequence-independent recog-
208
nition of amphiphilic a-helices, Trends Biochem. Sci.,
15, 59, 1990.
133. O’Neil, K. T., Wolfe, H. R., Jr., Erickson-
Viitanen, S., and De Grado, W. F., Fluorescence
properties of calmodulin-binding peptides reflect al-
134. Oh, S.-H. and Roberts, D. M., Analysis of the state
of post-translational calmodulin methylation in de-
veloping pea plants, Plant Physiol., 93, 880, 1990.
135. Oh, S.-H., Steiner, H.-Y., Dougall, D. K., and
Roberts, D. M., Modulation of calmodulin levels,
calmodulin methylation, and calmodulin binding pro-
teins during carrot cell growth and embryogenesis,
Arch. Biochem. Biophys., 297, 28, 1992.
136. Ohya, Y. and Anraku, Y., Functional expression of
chicken calmodulin in yeast, Biochem. Biophys. Res.
Commun., 158, 541, 1989.
137. Palme, K., Diefenthal, T., Hesse, T., Nitschke,
K., Campos, N., Feldwisch, J., Garbers, C., H e ,
F., Schwonke, S., and Schell, J., Signalling ele-
ments in higher plants; identification and molecular
analysis of an auxin-binding protein, GTP-binding
regulatory proteins and calcium sensitive proteins, in
Signal Molecules in Plants and Plant-Microbe Inter-
actions, NATO AS1 Series Vol. H 36, Lugtenberg,
B. J. J., Ed., Springer-Verlag, Berlin, 1989, 71.
138. Pantoja, O., Gelli, A., and Blumwald, E., Voltage-
dependent calcium channels in plant vacuoles, Science,
255, 1567, 1992.
139. Pausch, M. H., Kaim, D., Kunisawa, R., Admon,
A., and Thorner, J., Multiple Ca2+/calmodulin-de-
pendent protein kinase genes in a unicellular eukary-
ote, EMBO J., 10, 1511, 1991.
140. Perera, I. Y. and Zielinski, R. E., Structure and
expression of the Arabidopsis CaM-3 calmodulin gene,
Plant Mol. Biol., 19, 649, 1992.
141. Perera, I. Y. and Zielinski, R. E., unpublished
results from Genbank.
142. Perez-hat, E., Narasimhan, M. L., Binzel, M. L.,
Botella, M. A., Chen, Z., Valpuesta, V., Bressan,
R. A., and Hasegawa, P. M., Induction of a putative
Ca2+-ATPase mRNA in NaC1-adapted cells, Plant
Physiol., 100, 1471, 1992.
143. Pical, C., Sandelius, A. S., Melin, P.-M., and
Sommarin, M., Polyphosphoinositide phospholi-
pase C in plasma membranes of wheat (Triticum aes-
tivum L.): orientation of active site and activation by
Ca2+ and MgZ+,Plant Physiol., 100, 1296, 1992.
144. Polya, G. M. and Chandra, S., Ca2+-dependent
protein phosphorylation in plants: regulation, protein
substrate specificity and product dephosphorylation,
in Current Topics in Plant Biochemistry and Physi-
ology, Vol. 9, Plant Protein Phosphorylation, Protein
Kinases, Calcium and Calmodulin, Randall, D. D.
and Belvins, D. G., Eds., University of Missouri,
Columbia, 1990, 164.
145. Poovaiah, B. W. and Reddy, A. S. N., Calcium
messenger system in plants, Crit. Rev. Plant Sci., 6,
47. 1987.
 146. Poovaiah, B. W., Reddy, A. S. N.,and McFadden,
J. J., Calcium messenger system: role of protein
phosphorylation and inositol phospholipids, Physiol.
Plant., 69, 569, 1987.
147. Poovaiah, B. W., MacFadden, J. J., and Reddy,
A. S. N., The role of calcium ions in gravity signal
perception and transduction, Physiol. Plant., 7 1, 401,
1987.
148. Poovaiah, B. W., Glenn, G. M., and Reddy,
A. S. N., Calcium and fruit softening: physiology
and biochemistry, Hort. Rev., 10, 107, 1988.
149. Poovaiah, B. W. and Reddy, A. S. N., Calcium
and signal transduction: the involvement of calcium-
dependent protein phosphorylation and turnover of
inositol phospholipids, in Inositol Metabolism in
Downloaded by [University of Illinois at Urbana-Champaign] at 13:00 10 March 2015
Plants, Morre, J., Loewus, F., and Boss, W., Eds.,
Wiley-Liss, New York, 1990, 335.
150. Poovaiah, B. W., Reddy, A. S. N., An, G., Choi,
Y. J., and Wang, Z. Q., Calmodulin gene expres-
sion and Ca2+/calmodulin-dependentprotein kinase
I1 in plants, in Progress in Plant Growth Regulation,
Karssen, C. M., VanLoon, L. C., and Vreugdenhil,
D., Eds., Kluwer Academic Publishers, Dordrecht,
The Netherlands, 1992, 691.
151. Price, B. D. and Calderwood, S. K., Ca2+ is es-
sential for multistep activation of the heat shock factor
in permeabilized cells, Mol. Cell. Biol., 11, 3365,
1991.
152. Putnam-Evans, C. L., Harmon, A. C., and
Cormier, M. J., Purification and characterization of
a novel calcium-dependent protein kinase from soy-
bean, Biochemistry, 29, 2488, 1990.
153. Randall, D. D. and Belvins, D. G., Eds., Current
Topics in Plant Biochemistry and Physiology. Vol.
9, Plant Protein Phosphorylation, Protein Kinases,
Calcium and Calmodulin, University of Missouri,
Columbia, 1990.
154. Ranjeva, R. and Boudet, A. M., Phosphorylation
of proteins in plants: regulatory effects and potential
involvement in stimulus/response coupling, Annu.
Rev. Plant Physiol., 38, 73, 1987.
155. Ranjeva, R., Carrasco, A., and Boudet, A. M.,
Inositol trisphosphate stimulates the release of cal-
cium from intact vacuoles isolated from Acer cells,
FEBS Lett., 230, 137, 1988.
156. Rasmussen, C. D. and Means, A. R., Calmodulin
is required for cell-cycle progression during G, and
mitosis, EMBO J., 8, 73, 1989.
157. Ratan, R. R., Shelanski, M. L., and Maxfield,
F. R., Transition from metaphase to anaphase is ac-
companied by local changes in cytoplasmic free cal-
cium in Pt K2 kidney epithelial cells, Proc. Natl.
Acad. Sci. U.S.A., 83, 5136, 1986.
158. Raz, V. and Fluhr, R., Calcium requirement for
ethylene-dependent responses, Plant Cell, 4, 1123,
1992.
159. Rayson, B. M., Ca2+regulatss transcription rate of
the Na+/K+-ATPase subunit, J. Biol. Chem., 266,
21335, 1991.
160. Reddy, A. S. N., McFadden, J. J., Friedmann,
M., and Poovaiah, B. W., Signal transduction in
plants: evidence for the involvement of calcium and
turnover of inositol phospholipids, Biochem. Bio-
phys. Res. Commun., 149, 334, 1987.
161. Reddy, A. S. N. and Poovaiah, B. W., Molecular
cloning and sequencing of an auxin-repressed cDNA
clone: correlation between fruit growth and repression
of the auxin-regulated gene, Plant Mol. Biol., 14,
127, 1990.
162. Reddy, A. S. N., Wang, Z. Q., Choi, Y. J., An,
G., Czernik, A. J., and Poovaiah, B. W., Cal-
modulin gene expression and calciudcalmodulin de-
pendent protein kinase I1 in plants, Proc. Cold Spring
Harbor Symp. Plant Signal Transduction, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY, 1991,
57.
163. Reddy, A. S. N., An, G., and Poovaiah, B. W.,
unpublished results.
164. Reddy, A. S. N., Takezawa, D., Fromm, H., and
Poovaiah, B. W., unpublished results.
165. Resendez, E., Jr., Attenello, J. W., Grafsky, A.,
Chang, C. S., and Lee, A. S., Calcium ionophore
A23187 induces expression of glucose-regulated genes
and their heterologous fusion genes, Mol. Cell. Biol.,
5 , 1212, 1985.
166. Rizzuto, R., Simpson, A. W. M., Brini, M., and
Pozzan, T., Rapid changes of mitochondria1 Ca2+
revealed by specifically targeted recombinant ae-
quorin, Nature, 358, 325, 1992.
167. Roberts, D. M., Rowe, P. M., Siegel, F. L., Lukas,
T. J., and Watterson, D. M., Trimethyllysine and
protein function. Effect of methylation and mutage-
nesis of lysine 115 of calmodulin on NAD kinase
activation, J. Biol. Chem., 261, 1491, 1986.
168. Roberts, D. M., Lukas, T. J., and Watterson,
D. M., Structure, function, and mechanism of action
of calmodulin, Crit. Rev. Plant Sci., 4, 31 1, 1986.
169. Roberts, D. M., Besl, L., Oh, S.-H., Masterson,
R. V., Schell, J., and Stacey, G., Expression of a
cahodulin methylation mutant affects the growth and
development of transgenic tobacco plants, Proc. Natl.
Acad. Sci. U.S.A., 89, 8394, 1992.
170. Roberts, D. M. and Harmon, A. C., Calcium-mod-
ulated proteins: targets of intracellular calcium signals
in higher plants, Annu. Rev. Plant Physiol. Plant
Mol. Biol., 43, 375, 1992.
171. ROUX,S. J. and Slocum, R. D., Role of calcium in
mediating cellular functions important for growth and
development in higher plants, in Calcium and Cell
Function, Vol. 3, Cheung, W. Y., Ed., Academic
Press, New York, 1982.
172. ROUX,S. J., Guo, Y., and Li, H., Characterization
of two calcium-dependent protein kinases implicated
in stimulus-response coupling in plants, in Current
Topics in Plant Biochemistry and Physiology, Vol.
9, Plant Protein Phosphorylation, Protein Kinases,
Calcium and Calmodulin, Randall, D. D. and Bel-
vins, D. G., Eds., University of Missouri, Columbia,
1990, 129.
173. Rowe, P. M., Wright, L. S., and Siegel, F. L.,
Calmodulin N-methyltransferase: partial purification
and characterization, J. Biol. Chem., 261,7060, 1986.
 174. Ryazanov, A. G., Shestakova, E. A., and Natapov,
P. G., Phosphorylation of elongation factor-2 by EF-
2 kinase affects rate of translation, Nature, 334, 170,
1988.
175. Ryazanov, R. Z. and Spirin, A. S., Phosphorylation
of elongation factor 2: a key mechanism regulating
gene expression in vertebrates, New Biol., 2, 843,
1990.
176. Sanders, D., Miller, A. J., Blackford, S., Brosnan,
J. M., and Johannes, E., Cytosolic free calcium
homeostasis in plants, in Current Topics in Plant
.
Biochemistrv and Phvsiolonv. Vol. 9. Plant Protein
"I,
Phosphorylation, Protein Kinases, Calcium and Cal-
modulin, Randall, D. D. and Belvins, D. G., ms.,
University of Missouri, Columbia, 1990, 20.
Downloaded by [University of Illinois at Urbana-Champaign] at 13:00 10 March 2015
177. Scheuerlein, R., Schmidt, K., Poenie, M., and
ROUX,S. J., Determination of cytoplasmic calcium
concentration in Dryopteris spores, Planta, 184, 166,
1991.
178. Schroeder, J. I. and Thuleau, P., Caz+channels in
higher plant cells, Plant Cell, 3, 555, 1991.
179. Schroeder, J. I. and Hagiwara, S., Repetitive in-
creases in cytosolic Ca2+ of guard cells by abscisic
acid activation of nonselective Ca2+-permeable chan-
nels, Proc. Natl. Acad. Sci. U.S.A., 87,9305, 1990.
180. Schumaker, K. S. and Sze, H., Calcium transport
into the vacuole of oat roots, J. Biol. Chem., 261,
12172, 1986.
181. Schumaker, K. S. and Sze, H., Inositol 1,4,5-tris-
phosphate releases Caz+ from vacuolar membrane
vesicles of oat roots, J . Biol. Chem., 262, 3944,
1987.
182. Schumaker, K. S. and Sze, H., Solubilization and
reconstitution of the oat root vacuolar H+/Ca2+ex-
changer, Plant Physiol., 92, 340, 1990.
183. Sheng, M., Thompson, M. A., and Greenberg,
M. E., CREB: a Caz+-regulatedtranscription factor
phosphorylated by calmodulin-dependent kinases,
Science, 252, 1427, 1991.
184. Sherbany, A. A., Parent, A. S., and Brosins, J.,
Rat calmodulin cDNA, DNA, 6, 267, 1987.
185. Shina, T. and Tazawa, M., Ca2+-activated C1-
channel in plasmalemma of Bitellopsis obtusa, J .
Mem. Biol., 99, 137, 1987.
186. Sikela, J. M. and Hahn, W. E., Screening an
expression library with a ligand probe: isolation and
sequence of a cDNA corresponding to a brain cal-
modulin-binding protein, Proc. Natl. Acad. Sci.
U.S.A., 84, 3038, 1987.
187. Stevenson, M. A. and Calderwood, S. K., Mem-
bers of the 70-kilodalton heat shock protein family
contain a highly conserved calmodulin-binding do-
main, Mol. Cell. Biol., 10, 1234, 1990.
188. Stratowa, C. and Rutter, W. J., Selective regula-
tion of trypsin gene expression by calcium and by
glucose starvation in a rat exocrine pancreas cell line,
Proc. Natl. Acad. Sci. U.S.A., 83, 4292, 1986.
189. Suzuki, K. and Ohno, S., Calcium activated neutral
protease - structure-function relationship and func-
tional implications, Cell Struct. Funct., 15, 1, 1990.
210
190. Thuleau, P., Graziana, A., Canut, H., and
Ranjeva, R., A 75-kDa polypeptide located primar-
ily at the plasma membrane of carrot cell-suspension
cultures is photoaffinity labeled by the calcium chan-
nel blocker LU 49888, Proc. Natl. Acad. Sci. U.S.A.,
87, 1oo00, 1990.
191. Tobimatsu, T., Kameshita, I., and Fujisawa, H.,
Molecular cloning of the cDNA encoding the third
polypeptide (g) of brain calmodulin-dependent pro-
tein kinase 11, J. Biol. Chem., 263, 16082, 1988.
192. Tobimatsu, T. and Fujisawa, H., Tissue-specific
expression of four types of rat calmodulin-dependent
protein kinax I1 mRNAs, J . Biol. Chem., 264, 17907,
1989.
193. Toda, H., Yazawa, M., Sakiyama, F., and Yagi,
K., Amino acid sequence of calmodulin from wheat
germ, J. Biochem., 98, 833, 1985.
194. Trewavas, A. J., Ed., Molecular and Cellular As-
pects of Calcium in Plant Growth and Development,
Plenum Press, New York, 1986.
195. Trewavas, A. J. and Blowers, D. P., Protein kinases
in the plasma membrane, in Current Topics in Plant
Biochemistry and Physiology, Vol. 9, Plant Protein
Phosphorylation, Protein Kinases, Calcium and Cal-
modulin, Randall, D. D. and Belvins, D. G., Uni-
versity of Missouri, Columbia, 1990, 153.
196. Trewavas, A. J. and Gilroy, S., Signal transduction
in plant cells, Trends Genet., 7, 356, 1991.
197. Veluthambi, K. and Poovaiah, B. W., Calcium-
promoted protein phosphorylation in plants, Science,
223, 167, 1984.
198. Vorherr, T., James, P., Krebs, J., Enyedi, A.,
McCormick, D. J., Penniston, J. T., and Carafoh,
E.. Interaction of calmodulin with the calmodulin
binding domain of the plasma membrane Ca2+pump,
Biochemistry, 29, 355, 1990.
199. Wang, M., Van Duijn, B., and Schram, A. W.,
Abscisic acid induces a cytosolic calcium decreases
in barley aleurone protoplasts, FEBS Lett., 278, 69,
1991.
200. Wang, Z. Q., Reddy, A. S. N., Czernik, A. J.,
and Poovaiah, B. W., unpublished results.
201. Watillon, B., Kettmann, R., BOXUS,Ph., and
Burny, A., Cloning and characterization of an apple
(Malus domestica L. Borkh) cDNA encoding a cal-
modulin-binding protein domain similar to the cal-
modulin-binding region of type I1 mammalian Ca2+/
calmodulin-dependent protein kinase, Plant Sci., 8 1,
227, 1992.
202. Watillon, B., Kettmann, R., BOXUS,Ph., and
Burny, A., Cloning and characterization of an apple
(Malus domestica L. Borkh) calmodulin gene, Plant
Sci., 82, 201, 1992.
203. Watillon, B., Kettmann, R., BOXUS,Ph., and
Burny, A., A calciudcalmodulin-binding serinel
threonine protein kinase homologous to the mam-
malian type I1 calciudcalmodulin-dependent protein
kinase is expressed in plant cells, Plant Physiol., 101,
1381, 1993.
 204.Wegner, M., Cao, Z., and Rosenfeld, M. G., Cal-
cium-regulated phosphorylation within the leucine
zipper of CIEBPP, Science, 256, 370, 1992.
205. White, B. A. and Bancroft, C., Regulation of gene
expression by calcium, in Calcium and Cell Func-
tion, Academic Press, New York, 1987, 109.
206. Widada, J. S., Asselin, J., Colote, S., Marti, J.,
Ferraz, C., Trave, C., Haiech, J., and Liautard,
J.-P., Cloning and deletion mutagenesis using direct
protein-protein interaction on an expression vector,
J. Mol. Biol., 205, 455, 1989.
207. Williams, D. A., Becker, P. L., and Fay, F. S.,
Regional changes in calcium underlying contraction
of single smooth muscle cells, Science, 235, 1644,
1988.
Downloaded by [University of Illinois at Urbana-Champaign] at 13:00 10 March 2015
208.Williamson, R. E. and Ashley, C. C., Free Ca2+
and cytoplasmic streaming in the alga, Chara, Nature,
296,647, 1982.
209.Wimmers, L. E., Ewing, N. N., and Bennett,
A. B., Higher plant Ca2+-ATPase:primary structure
and regulation of mRNA abundance by salt, Proc.
Natl. Acad. Sci. U.S.A.. 89,9205, 1992.
210.Yang, W.,Burkhart, W., Cavallius, J., Merrick,
W. C., and BOSS,W. F., Purification and charac-
terization of a phosphatidylinositol4-kinaseactivator
in carrot cells, J. Biol. Chem., 268, 392, 1993.
21 I Ziegenhagen, R. and Jennissen, H. P., Plant and
fungus calmodulins are polyubiquinated at a single
site in a Caz+-dependentmanner, FEBS Lett., 273,
253, 1990.
212. Zielinski, R. E., Ling, V., and Perera, I., Structure
and expression of genes encoding calcium-modulated
proteins in higher plants, in Current Topics in Plant
Biochemistry and Physiology, Vol. 9, Plant Protein
Phosphorylation, Protein Kinases, Calcium and Cal-
modulin, Randall, D. D. and Belvins, D. G., Eds.,
University of Missouri, Columbia, 1990, 141.
213.Zimmer, W. E., Schloss, J. A., Silflow, C. D.,
Youngblom, J., and Watterson, D. M., Structural
organization, DNA sequence, and expression of the
calmodulin gene, J. Biol. Chem., 263,19370,1988.
214.Takeda, T. and Yamamoto, M., Analysis and in
vivo disruption of the gene coding for calmodulin in
Schizosaccharomyces pombe, Proc. Natl. Acad. Sci.
U.S.A., 84,3580, 1987.
211