Download The alphabet soup of plant intracellular signalling: enter cyclic

419
The alphabet soup of plant intracellular signalling: enter cyclic
nucleotides
Richard Walden
Recent work reveals a role for cyclic nucleotides as secondary
signalling molecules in a variety of signal transduction
pathways in plants. Evidence is accumulating that cGMP is
involved in signalling during photomorphogenesis and that
cADP-ribose triggers the release of sequestered Ca2+ during
the response of plant cells to abscisic acid. Though more
tentative, cAMP has been proposed as playing an important
role in ion channel activity and cell cycle progression. Taken
together, a picture emerges of differing signalling pathways,
possibility interacting with each other, acting on an array of
developmental processes.
Addresses
Department of Plant Breeding and Biotechnology, Horticulture
Research International, East Malling, West Malling, ME19 6BJ, UK and
Carnegie Institution of Washington, Department of Plant Biology, 260
Panama Street, Stanford, CA 94305, USA;
e-mail: [email protected]
Current Opinion in Plant Biology 1998, 1:419–423
http://biomednet.com/elecref/1369526600100419
© Current Biology Ltd ISSN 1369-5266
Abbreviations
ABA
abscisic acid
CADPR cyclic ADP-ribose
EST
expressed sequence tag
myo-inositol 1,4,5 triphosphate
IP3
NAADP nicotinic acid adenine dinucleotide phosphate
RyR
ryanodine receptors
Introduction
Intracellular signalling relies on the perception of a signal
at the plasmamembrane by a receptor, activating the formation of a secondary signal within the cytosol which
ultimately modifies activity of an effector molecule. Such
signalling has three purposes; first, it converts the primary
signal into a form recognised by an effector; second, it
allows signal amplication; and third, depending on the
physiological status of the cell, it allows signal damping. In
nonhigher-plant eucaryotic systems, pathways of intracellular signalling are well established. Although plant
biologists often draw parallels with animal systems, our
knowledge of secondary signalling molecules in plants is
fragmentary, with the possible exception of Ca2+ ions.
Often the difficulty has been a lack of knowledge of the
receptors and effectors in such signalling processes.
Recently a variety of novel molecular genetic approaches
have been adopted to address not only whether a specific
signalling molecule is active in plant cells but also the
process in which such molecules may be involved. A picture is beginning to develop of several secondary
signalling molecules triggering a variety of processes.
Though signalling may be specific, there is potential for
interaction between differing pathways, thus resulting in a
complex signalling network. Hence, one is confronted
increasingly by a confusing array of signalling molecules,
their biosynthetic enzymes and effectors, often referred to
by abbreviation. Here I describe some of the recent work
that has revealed that cyclic nucleotides are involved in
secondary signalling in plants and attempt to make some
sense of what might be seen as the emerging alphabet
soup of intracellular signalling in plants.
Calcium signalling
The first, and many would say the most important, ingredient of the intracellular signalling alphabet soup is
calcium (Trewavas and Malhó, pp 428–433). Ca2+ ions act
as a secondary signalling molecule widely in eucaryotes. In
plants, Ca2+ has been implicated in intracellular signalling
directly, or indirectly in the action of a large number of
phytohormones with the interesting exception of ethylene
[1]. What makes Ca2+ intriguing as a signalling molecule is
that it is not just its presence or absence that provides signalling capability, rather the local Ca2+ concentration
experienced by its effector. Following stimulation, local
cytoplasmic Ca2+ concentrations can be raised many fold
and slowly decay, or they can oscillate or pass in wave form
[2,3,4]. These patterns of accumulation likely play a role in
signalling specificity and require that internal local concentration of free Ca2+ ions be exquisitely controlled.
There are three likely means of doing this. Ca2+ can be
actively pumped into, or from, the cytosol by pumps located in the vacuole [5] and the plasma membranes [6]. Ca2+
may be sequestrated in organelles. Finally Ca2+ may bind
a variety of proteins, for example calmodulin, changing
their activity, or the protein–Ca complex might simply act
as a storage form of the ion. Ultimately, changes in Ca2+
level elicit a response, for example changes in gene
expression [7]. Sequestered Ca2+ can be loaded into the
cytosol in response to the action of one of three secondary
signalling molecules: myo-inositol 1,4,5 triphosphate (IP3),
cyclic ADP-ribose (cADPR) and nicotinic acid adenine
dinucleotide phosphate (NAADP) [8,9] (Figure 1).
Secondary signalling in photomorphogenesis
Often, studies in secondary signalling have been hampered by lack of an appropriate, easily manipulatable,
experimental system. Neuhaus, Chua and co-workers have
elegantly overcome this by use of the tomato phytochrome
A lacking aurea mutant for microinjection studies
[10,11,12]. The concept is straightforward. Inject
hypocotyl cells of dark grown seedlings with DNA containing a promoter that responds to a specific stimulus
linked to a marker gene either by itself, or in the presence
of other compounds. Subject the injected hypocotyls to an
external stimulus and measure expression of the marker
gene after two days. In ground breaking work these
420
Cell signalling and gene regulation
Figure 1
NADP
NAADP
(+)
Ca2+
Extracellular
signal
cADPR (+)
NAD+
RyR
Ca2+
Ca2+
DAG
Receptor
IP3
(+)
Protein
phosphorylation
IP3R
Regulation of
cellular function
Ca2+
Phospholipase C
Ca2+
pumps/channels
Current Opinion in Plant Biology
authors demonstrated that phytochrome action can be
mimicked by Ca2+ and cGMP and that possibly one or
more heterotrimeric G proteins are involved [10].
Photomorphogenesis can be divided into three processes:
those triggered by Ca2+ (expression of chlorophyll a,bbinding proteins and partial chloroplast development),
those triggered by cGMP (chalcone synthase and anthocyanins) and those triggered by both (ferredoxin NADP+
oxidoreductase and full chloroplast maturation). These
pathways also apparently cross regulate each other so that
high levels of cGMP down regulate the Ca2+ pathways and
vice versa [11]. The sequences of the target promoters
responsive to cGMP and Ca2+ have been identified [12].
Significantly, the same Ca2+/cGMP pathway can also down
regulate activity of the asparagine synthase promoter
[13••]. More recent work, this time using dark adapted
cells from a soybean suspension culture maintained in light
and sucrose, has shown that the down regulation of chalcone synthase expression by Ca2+ as a phytochrome
response can be reversed in UV light. The phytochrome
response precedes the UV response suggesting that the
apparent antagonistic action of Ca2+ in the cell is possible
because the two processes are temporally separated [14•].
Secondary signalling in the ABA response
A characteristic of secondary messenger signalling is that
subsequent signalling events include protein phosphorylation involving the action of protein kinases and
phosphatases. The abscissic acid (ABA) insensitive
Arabidopsis mutants ABI1 and ABI2 are changed in a serine-threonine protein phosphatase with calcium binding
domains [15,16,17]. Building on this, the effect of expression of genes encoding different Ca2+-dependent protein
kinases and phosphatases has been tested directly in maize
protoplasts [18]. In elegantly simple experiments, the
Secondary signalling via cyclic nucleotides.
Ca2+ release from intracellular stores can
occur by one of three processes. Following
reception of an extracellular signal, inositol
triphosphate (IP3) can be released from the
plasma membrane, along with diacylglycerol
(DAG), by the action of phospholipase C and
IP3 stimulates Ca2+ release into the cytoplasm
via an IP3 receptor/channel (IP3R). cyclic
ADP-ribose (cADPR) is synthesised by the
action of ADP-ribosyl cyclase using oxidised
nicotinamide adenine dinucleotide (NAD+) as
a substrate. cADPR acts on the Ca2+ release
channel, the ryanodine receptor (RyR).
Nicotinic acid adenine dinucleotide phosphate
(NAADP), yet to be characterised as a
signalling molecule in plants, can be
generated from a common pyridine adenine
dinucleotide redox cofactor (NADP). In
addition, intracellular levels of Ca2+ can be
controlled by a variety of channels and pumps.
expression of an ABA responsive promoter, HVA1, linked
to either the jelly fish fluorescent protein gene or the
luciferase gene was tested following cotransfection with
genes encoding either a palette of protein kinases or of
phosphatases. Specific constitutive kinase expression can
trigger expression of the HVA1 promoter in the absence of
ABA or stress signals, whereas a constitutively expressed
protein phosphatase 2C blocks the response. Significantly,
phosphatase mutants act in a dominant manner to inhibit
expression, consistant with the dominant ABI1/2 phenotype. This might be explained by the mutant protein
titrating out its molecular partners and allows molecular
analysis of the interactions of the phosphatase with partner
proteins [19••].
It has been known for some time that IP3 mobilises Ca2+
from plant cell vacuoles [20] and more recently it has been
shown that, as in animals, cADPR releases sequestered
Ca2+ via channels co-localising with the IP3 gated channels
[21]. Pharmaceutical analysis supports the expectation that
cADPR activated Ca2+ release is mediated by the activation of ryanodine sensitive Ca2+ channels (ryanodine
receptors [RyR]) [22]. Suspicion that cADPR might be
involved in the ABA response [23] has been convincingly
confirmed by recent work from the Chua laboratory, once
again using hypocotyl microinjection [24••]. In this case,
the target promoters were a desiccation sensitive promoter
(rd29A) and a promoter from a cold inducible gene (kin2).
Expression from both promoters is induced by ABA treatment as well as the injection of either Ca2+ and cADPR in
the absence of ABA. Injection of ADP-ribosyl cyclase, the
enzyme responsible for cADPR formation from NAD+,
also triggers expression of both promoters in the absence of
ABA. The cation chelator EGTA blocks ABA induced
expression as well as that induced by Ca2+, ADP-ribosyl
cyclase and cADPR suggesting that Ca2+ acts downstream
The alphabet soup of plant intracellular signalling: enter cyclic nucleotides Walden
of cADPR in the signalling pathway. This notion is supported by the finding that injection of 8NH2–cADPR, a
competitive inhibitor of cADPR, blocked induction by
ABA, the cyclase, cADPR, but not Ca2+. In addition, injection of NADase (removing the substrate for cADPR
synthesis) blocks ABA induction.
In the light of the work with maize protoplasts, it might not
be a surprise that the phosphatase inhibitor okadaic acid
activates gene expression from the ABA responsive promoters, whereas the kinase inhibitor K252a blocks it. The
versatility of the microinjection system, however, is
demonstrated by finding that okadaic acid induced expression is blocked by either EGTA, or 8NH2–cADPR placing
the phosphatase requirement upstream of cADPR production and Ca2+ release. On the other hand, K252a blocks
ABA independent expression induced by okadaic acid,
cADPR and Ca2+ placing the required kinase activity
downstream of Ca2+ release. The proposition that cADPR
triggered Ca2+ release plays a role in ABA induction of
gene expression gains further support using Arabidopsis
transformed with the kin2 promoter linked to luciferase.
Here the time course of gene expression correlates with
increases in cADPR levels.
Where is the specificity of the Ca2+ response? Ca2+ also triggered photomorphogenesis in the same experimental
system. Does triggering of the cab (chlorophyll a/b binding
protein) promoter in the phyA (phytochrome A) system
share the same signalling pathway as the ABA induction of
rd29A and kin2 promoters? Apparently not— in important
control experiments, both rd29A and kin2 promoters were
not induced by phyA. Conversely, the cab promoter is not
induced by cADPR. Tissue specific characteristics, or cellular competance has been argued as playing a role in
specificity of Ca2+ signalling [25]; however, tissue specificity is hard to argue in this case. One wonders if specificity
might be provided by the mode of Ca2+ release. With this
in mind, co-injection of IP3 also activates ABA inducible
genes in the absence of ABA but not the expression of the
cab promoter. The IP3 specific blocker heparin does not
block ABA induced gene expression suggesting that IP3
might be involved in a secondary ABA response [24].
cADPR as a signalling molecule may be widely distributed
throughout the plant kingdom. It has been described as
oscillating in levels in the unicellular Euglena during the
cell cycle with maximal levels being observed immediately before cell division [26].
cAMP: the outsider of plant secondary
signalling
Compared with other secondary signalling molecules,
cAMP has had a troubled history in plant biology. Many
workers have attempted to demonstrate a role for cAMP in
plants, however, results have not been unequivocal, largely because of the apparently low levels of cAMP in plants
which may border on the artifactual [27,28].
421
Using whole cell patch clamping of bean mesophyll protoplasts it has been found that cAMP can increase outward
K+ current in a dose dependent manner. This effect is
mimicked by the catalytic subunit of a mammalian protein
kinase A (PKA) in the absence of cAMP [29]. More recently, excitement was stirred by a report of the isolation of a
gene from tobacco with limited similarity to adenyl cyclase
from S. pombe whose expression was able to activate protoplast division and it was suggested that cAMP might play a
role in auxin triggered cell division [30]. The protoplast
assay used in this case is currently under review, however,
and until independently confirmed this report must be
treated with caution. More significant, however, may be
the recent finding that cAMP levels may oscillate in suspension cells during the cell cycle [31]. Here, using a
tobacco suspension cell system that can be synchronised
with relative ease, it was shown that there are transient
peaks of cAMP levels in S and G1 phases of the cell cycle.
This suggests a role for cAMP in cell cycle progression,
though, as it stands, the data is only correlative. It would be
interesting to check the effect of cAMP of the activity of
components of cell cycle progression or their expression. A
related observation might be relevant; expression of the
cauliflower mosaic virus (CaMV) 35S RNA promoter in
yeast is dependent on cAMP [32]. A general transcription
factor likely to bind to a portion of the CaMV 35S RNA
promoter contains a region similar to the mammalian
cAMP responsive element binding (CREB) factor [33].
Expression of the 35S RNA promoter in this tobacco cell
line correlates with the changes of cAMP levels reported
[34]. A coincidence? Possibly, though one that could be
tested relatively simply.
Secondary signalling in the post Gutenburg era
Advances in plant expressed sequence tag (EST) and
genomic DNA sequencing raises the possibility that genes
encoding elements of signalling pathways might be identified by sequence similarity with genes from other systems
before the pathway itself has been defined either biochemically, or by molecular genetics in plants. A foretaste
is provided by a quick scan of the TIGR Arabidopsis gene
index [35] which reveals four hits when searched for RyR,
251 with protein kinase A and 217 with protein kinase C.
Naturally, DNA sequence alone does not suffice to implicate a specific gene product in a specific signalling
pathway, and there is a need to sort out which gene product may be involved in which pathway. Nevertheless,
computer searches can provide valuable pointers as
demonstrated by the recent cloning of a gene encoding a
seven membrane span receptor-like protein [36••,37].
In this case PCR was used to isolate the GCR1 gene encoding a seven membrane span receptor-like protein with
greatest similarity to CAR1, the Dictyostelium cAMP receptor. Southern analysis reveals GCR1 to be a single copy gene
in Arabidopsis. Arabidopsis transformants expressing antisense GCR1 displayed the dainty phenotype: reduced
cotyledon and leaf expansion as well as the production of a
422
Cell signalling and gene regulation
single flowering stem at the five–eight leaf stage [36••].
Roots of dainty seedlings have reduced sensitivity to higher
levels of cytokinins compared with plants transformed with
the vector lacking the GCR1 antisense gene. The dainty
phenotype is reminiscent of the Arabidopsis cytokinin resistant mutant cyr1 [38], though does not appear to be allelic
to it. The authors suggest GCR1 is involved in cytokinin
signalling. The question of course is how direct, or indirect,
this involvement might be. Previously, CKI1 has been isolated by activation T-DNA tagging followed by selection
for calli developing in a cytokinin related manner even
though cytokinin was absent from the culture media [39].
CKI1 encodes a two component receptor kinase-like protein. Clearly, it will be important to check whether either
CKI1, or GCR1 acts as a cytokinin receptor. In the meantime one wonders whether GCR1, true to it’s similarity to
CAR1, is actually a cAMP receptor? This would raise the
intriguing possibility of cAMP acting in paracrine signalling
in plants as in Dictostelium.
Conclusions
Cyclic nucleotides are now established as secondary signalling molecules in plants with cGMP playing a role in
photomorphogenesis and cADPR in ABA signalling.
cAMP remains the outsider of plant biology, but biochemical evidence suggests a role in ion channel function and
cell cycle progression. Most probably it is only a matter of
time until NAADP is added to this list.
Ca2+
release into the cytosol appears to be an integral part
of signalling in a number of developmental processes; this
raises a question of specificity of action. The requirement
for specificity has been elegantly demonstrated in the
experiments involving microinjection where differing signals apparently result in Ca2+ release, which in turn
culminates in a differential response in the same cell type.
Understanding the mechanism of this differential response
is one of the greatest challenges arising from this work.
Probably it involves mechanism of release, the mode of
intracellular local Ca2+ accumulation (i.e. single burst, wave
form or oscillation) as well as subcellular compartmentation.
The work described here provides important pieces of the
puzzle of signal transduction in plant cells, yet there are
still important gaps in our knowledge. Many vital components of these signalling pathways remain to be
characterised — these include the receptors involved at
one end of the signalling pathway and the effectors at the
other end. It may be significant that mutational analysis
has yet to provide a means of isolating these, particularly
the receptors, and the question arises as to whether mutational screens have been designed in an appropriate
manner to isolate genes, or whether functional redundancy
is involved. As shown by the characterisation of GCR1,
genomic and EST sequencing are likely to provide us with
genes encoding candidates for components of signalling
pathways, but the trick will be to link these genetically and
biochemically to a particular signalling process.
One of the refreshing aspects of recent work is that novel
experimental systems, for example protoplasts and the
technique of micro-inoculation, have been used successfully to dissect secondary signalling. Often, certainly in the
case of phytohormone signalling, experimental systems
which may be generally difficult to manipulate have been
adopted. On the face of it these new experimental
approaches appear robust and amenable to manipulation
though the results obtained with them need to extrapolated to other, more general systems of studying
phytohormone responses. Indeed, it does not take a lot of
imagination to suspect that, with appropriate promoter
constructs, these systems could be effectively used to
study signalling pathways used by other phytohormones of
which we know so little, such as auxin and cytokinin.
The components of the intracellular soup of secondary signalling that are currently emerging allow for an interaction
between differing pathways, for example the Ca2+ and
cGMP pathways involved in photomorphogenesis. In considering that in animal systems such pathways are involved
in a variety of diverse processes such as apoptosis and
response to free radicals such as NO [8] it can be expected
that in the near future more examples of the spectrum of
events in the plant cell in which cyclic nucleotides are
involved will emerge.
Acknowledgements
Thanks to members of the Walden lab, Max-Planck Institut fur
Zuchtungforschung Cologne, particularly Carla Schommer, Tine Rausch
and Elke Bongartz for their support.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Bethke PC, Gilroy S, Jones RL: Calcium and plant hormone action.
In Plant Hormones. Edited by Davies PJ. Dordrecht: Kluwer Academic;
298-317.
2.
McAinsh MR, Webb AAR, Taylor JE, Hetherington A: Stimulus
induced oscillations in guard cell free calcium. Plant Cell 1995
7:1207-1219.
3.
Ehrhardt DW, Wais R, Long SR: Stimulus induced oscillations in
guard cell cytosolic free calcium. Cell 1996 85:673-681.
4.
Franklin-Tong VE, Droback BK, Allan AC, Watkins PAC, Trewavas AJ:
Growth of pollen tubes of Papaver rhoeas is regulated by a slow
moving calcium wave propagated by inositol 1,4,5-triphosphate.
Plant Cell 1996 8:1305-1321.
5.
Allen GJ, Sanders D: Two voltage-gated, calcium release channels
co-reside in the vacuolar membrane of broad bean guard cells.
Plant Cell 1995 6:685-694.
6.
Dainese P, James P, Baldon B, Carafoli E: Subcellular and tissue
distribution, partial purification and sequencing of calmodulin
stimulated Ca2+-transporting ATPases from Barley Hordeum
vulgare (L.) and tobacco Nicotiana tabacum. Eur J Biochem 1997
244:31-38.
7.
Hardingham GE, Chawla S, Johnson CM, Bading H: Distinct
functions of nuclear and cytoplasmic calcium in control of gene
expression. Nature 1997 385:260-265.
8.
Hancock JT: Cell Signalling 1997, Harlow, Essex; Longman.
9.
Dousa TP, Chini EN, Beers KW: Adenine nucleotide diphosphates:
emerging second messengers acting via intracellular Ca2++
release. Am J Physiol 1996 40:1007-1024.
The alphabet soup of plant intracellular signalling: enter cyclic nucleotides Walden
10. Bowler C, Neuhaus G, Yamagata H, Chua N-H: Cyclic GMP and
calcium mediated phytochrome phototransduction. Cell 1994,
77:73-81.
11. Bowler C, Yamagata H, Neuhaus G, Chua N-H: Phytochrome signal
transduction pathways are regulated by reciprocal control
mechanisms. Genes Dev 1994, 8:73-81.
12. Wu Y, Hiratsuka K, Neuhaus G, Chua N-H: Calcium and cGMP
target distinct phytochrome responsive elements. Plant J 1996,
10:1149-1154.
13. Neuhaus G, Bowler C, Hirasuka K, Yamagata H, Chua N-H:
•• Phytochrome regulated repression of gene expression requires
calcium and cGMP EMBO J 1997, 16:2554-2564.
In addition to up regulating gene expression the Ca2+/cGMP system, it is
shown that this system can down regulate gene expression. This paper is
also important because loss and gain of function experiments identify a 17
basepair cis element within the promoter of the asparagine synthase gene
necessary and sufficient for this down regulation. This element contains a
sequence motif present in the phytochrome A promoter: TGGG.
14. Frohnmeyer H, Bowler C, Zhu J-K, Yamagata H, Schäfer E, Chua N-H:
•
Different roles for calcium and calmodulin in phytochrome- and
UV-regulated expression of chalcone synthase. Plant J 1998,
13:763-772.
Important in that this work suggests that differential responses to changes
in Ca levels may be specified by temporal changes in Ca flux.
15. Leung J, Bouvier-Durand M, Morris P-C, Guerrier D, Chefdor F,
Giraudat J: Arabidopsis ABA response gene ABI1: features of a
calcium-modulated protein phosphatase. Science 1994,
264:1448-1452.
16. Meyer K, Leube MP, Grill E: A protein phosphatase 2C involved in
ABA signal transduction in Arabidopsis thaliana. Science 1994,
264:1452-1455.
17.
Leung J, Merlot S, Giraudat J: The Arabidopsis ABSCISSIC ACID
INSENSITIVE 2 (ABI2) and ABI1 genes encode homologous
protein phosphatase 2C involved in abscissic acid signal
transduction. Plant Cell 9:759-771.
423
24. Wu Y, Kuzma J, Marechal E, Graeff R, Lee HC, Foster R, Chua N-H:
•• Abscissic acid signalling through cyclic ADPribose in plants.
Science 1997, 278:2126-2130.
Once again the elegant simplicity of microinjection throws light on a
signalling pathway, this time that triggered by ABA. The microinjection
experiments are backed up using Arabidopsis engineered to contain an
ABA-responsive marker construct and it is shown that increases in cADPR
mimic expression of the marker gene.
25. McAinsh MR, Hetherington AM: Encoding specificity in Ca2+
signalling systems. Trends Plant Sci 1998, 3:32-36.
26. Masuda W, Takenaka S, Inageda K, Nishina K, Takahashi K, Katada T,
Tsuyama S, Inui H, Miyatake K, Nakano Y. Oscillation of ADP ribosyl
cyclase activity and function of cADP ribose in a unicellular
organism, Euglena gracilis. FEBS Letts 1997, 405:104-106.
27.
Spiteri A, Viratelle OM, Raymond P, Rancillac M, Labousse J, Pradet
A: Artifactual origins of cyclic AMP in higher plant tissues. Plant
Physiol 1989, 91:624-628.
28. Assmann S: Cyclic AMP as a second messenger in higher plants:
status and future prospects Plant Physiol 1995, 108:885-889.
29. Li W, Luan S, Schreiber SL, Assmann SM: Cyclic AMP stimulates K+
channel activity in mesophyll cells of Vicia faba L. Plant Physiol
1994, 106:957-961.
30. Ichikawa T, Suzuki Y, Czaja I, Schommer C, Lessnick A, Schell J,
Walden R: Identification and role of adenyl cyclase in auxin
signalling in higher plants. Nature 1997, 390:698-701.
31. Ehsan H, Reichheld J-P, Roef L, Witters E, Lardon F, Bockstaele DV,
Van Montagu M, Inze D, Van Onckelon H: Effect of indomethanacin
on cell cycle dependent cyclic AMP fluxes in tobacco BY-2 cells.
FEBS Letts 1998, 422:165-169.
32. Rüth J, Hirt H, Schweyen RJ: The cauliflower mosaic virus 35S
promoter is regulated by cAMP in Saccharomyces cerevisiae. Mol
Gen Genet 1992, 235:365-372.
33. Katagiri F, Lam E, Chua N-H: Two tobacco DNA-binding proteins with
homology to the nuclear factor CREB. Nature 1989, 340:727-730.
18. Sheen J: Ca2+-dependent protein kinases and stress signal
transduction in plants. Science 1996, 274:1900-1902.
34. Nagata T, Okada K, Kawazu T, Takebe I: Cauliflower mosaic virus
35S promoter directs S phase specific expression in plant cells.
Mol Gen Genet 1987, 207:242-244.
19. Sheen J: Mutation analysis of protein phosphatase 2C involved in
•• abscissic acid signal transduction in higher plants. Proc Natl Acad
Sci USA 1998, 95:975-980.
In an elegant demonstration of the power of the protoplast system, a
molecular analysis is used to assay the functional requirements of the
protein phosphatase 2C in the ABA response leading to gene expression.
35. The Institute for Genome Research Arapidopsis Gene index on the
World Wide Web URL: http://www.tigr.org/tdb/agi/index.html.
20. Alexandre J, Lassalles JP, Kado RT: Opening of Ca2+ channels in
isolated red beet root vacuole membrane by inositol 1,4,5triphosphate. Nature 1990, 343:567-570.
21
Allen GJ, Muir SR, Sanders D: Release of Ca2+ from individual
plant vacuoles by both InsP3 and cyclic ADP ribose. Science
1996, 268:735-737.
36. Plakidou-Dymock S, Dymock D, Hooley R: A higher plant seven
•• membrane receptor that influences sensitivity to cytokinins.
Current Biol 1998, 8:315-324.
PCR is used to isolate GCR1 from Arabidopsis, a seven membrane receptorlike gene with greatest similarity to CAR1 a cAMP receptor of Dictyostelium.
The gene is one of a receptor-like gene family in Arabidopsis. Antisense
expression of GCR1 results in a decreased sensitivity to cytokinins.
37.
Josefsson LG, Rask L: Cloning of a putative G-protein coupled
receptor from Arabidopsis thaliana. Eur J Biochem 1997, 249:415420.
22. Muir SR, Sanders D: Pharmacology of Ca2+ release from red beet
microsomes suggests the presence of ryanodine receptor
homologs in higher plants. FEBS Letts 1996, 395:39-42.
38. Deikman J, Ulrich M: A novel cytokinin-resistant mutant of
Arabidopsis with abbreviated shoot development. Planta 1995,
195:440-449.
23. Leckie CP, Mcainsh MP, Hetherington AR: cADPR and stomatal
closure Plant Physiol 1997, 114:272.
39. Kakimoto T: CKI1, a histidine kinase homolog implicated in
cytokinin signal transduction. Science 1996, 274:982-985.