Calcium-mediated signal transduction in plants: A perspective on the role of Ca2+ and CDPKs during early plant development

J. Plant Physiol. 158. 1237 – 1256 (2001)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp

Review Calcium-mediated signal transduction in plants: A perspective on the role of Ca2 + and CDPKs during early plant development Veena S. Anila, K. Sankara Rao* Department of Biochemistry, Indian Institute of Science, Bangalore-560 012, India Received January 29, 2001 · Accepted May 9, 2001

Summary In plants, calcium acts as a second messenger in the regulation of a variety of physiological and metabolic processes. For signal transduction via calcium, transient and often local elevations in the level of cytosolic Ca2 + must occur. This requires a well-controlled co-operation of tightly regulated Ca2 + -channels and active Ca2 + -transporters such as primary Ca2 + -ATPases and the H + /Ca2 + antiporters found both at the plasma membrane and endomembranes. Downstream events in calcium signaling involve Ca2 + -binding proteins and protein kinases that sense, amplify and transduce the Ca2 + -signal further downstream. This review gives an overview of the Ca2 + -messenger system and its components in plants. Although endosperm development, embryogenesis and germination are dynamic and intriguing developmental processes in the life cycle of plants, very little is known about the signaling events that regulate them. Recent observations suggest the involvement of Ca2 + -mediated signaling and Ca2 + -dependent protein kinases during these early-developmental processes in plants. A perspective of Ca2 + -signaling during seed development and germination is presented. Key words: embryogenesis – Ca2 + -second messenger system – Ca2 + -sensor proteins – calmodulin – calmodulin-like domain protein kinase – Ca2 + -dependent protein kinase – CaM kinase – germination – signal transduction Abbreviations: CDPK calcium-dependent protein kinase. – CaM calmodulin. – CBPs CaM binding proteins. – EGTA ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid. – W7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide

Introduction Plants exhibit high levels of phenotypic and physiological plasticity as a response to a multitude of environmental and a

Current address: National Centre for Biological Sciences, UAS-GKVK Campus, Bangalore 560065, India * E-mail corresponding author: [email protected]

hormonal signals. Plants can adapt and survive under stressful conditions, such as drought, salinity, change in soil pH, and anoxia caused by water-logging and mechanical stress brought about by strong winds etc. They also have the ability to synchronize developmental programs with the seasons and defend themselves against fungal, viral and bacterial infections. Innumerable examples of ‹intelligent› plant-responses indicate that plants have evolved mechanisms by 0176-1617/01/158/10-1237 $ 15.00/0

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Veena S. Anil, K. Sankara Rao

Table 1. Various stimuli that induce [Ca2 + ]cyt elevation in plants. Signal

Response

Reference

NOD factors Red light Abscisic acid, Oxidative stress, cold Hypoosmotic shock Salinity Touch Fungal elicitors Cold Heat shock Oxidative stress Gibberellin Anoxic stress cGMP and cAMP Transfer to darkness Pollen tube growth Increased osmoticum and auxin withdrawal Gibberelic acid

Root hair curling Photomorphogenesis Stomatal closure Osmoadaptation Drought resistance Growth retardation Phytoalexin synthesis Cold adaptation Thermotolerance Free radical scavenging α-Amylase secretion Anaerobic gene expression Swelling of protoplast Circadian rythem Pollen tube orientation Somatic embryogenesis α-amylase synthesis in barley aleurone

Erhardt et al. (1996) Shacklock et al. (1992) Mc Ainash et al. (1990); Allen et al. (2000) Taylor et al. (1996); Cessna et al. (1998) Knight et al. (1997) Knight et al. (1991) Knight et al. (1991) Knight et al. (1996); Plieth (1999); Plieth et al. (1999) Gong et al. (1998) Price et al. (1994) Bush and Jones (1988) Subbaiah et al. (1994) Volotovski et al. (1998) Johnson et al. (1995) Malho and Trewavas (1996) Anil and Sankara Rao (2000) Bethke et al. (1995); Gilroy and Jones (1992)

which they can perceive, transduce, analyze, and integrate a multitude of stimuli, and ultimately respond in the most appropriate manner. Plant cell biologists now realize that complete understanding of this cellular process could have far-reaching implications in agriculture, biotechnology, and forestry. A simplified scheme of the well-understood signal/response coupling mechanism in animals, a prototype in deciphering signaling events in less-well-understood systems, is as follows: 1. Perception of the stimulus by membrane-bound surface receptors or intracellular receptors 2. Activation of certain membrane functions such as opening of ion channels (Levitan 1985), activation of G-proteins, production of second messengers by hydrolysis of membrane components, such as phosphoinositides (Berridge 1984, Hokin 1985), and stimulation of enzymes, such as adenylate cyclase (Lefkowitz et al. 1983) 3. Increase in the cytosolic concentration of second messengers 4. Activation of second messenger-dependent enzymes, especially protein kinases 5. Phosphorylation of proteins and amplification of the stimulus 6. Dephosphorylation that results in turning off the stimulus and returning to the resting situation. Although our knowledge of plant signal transduction remains incomplete, the identification of components of the abovementioned scheme, such as perception-receptors (Fairchild and Quail 1998), ion channels (White 2000), G proteins (Jones et al. 1998), second messengers (Sanders et al. 1999), second messenger-binding proteins, and protein kinases (Roberts 1993, Watillon et al. 1995, Patil et al. 1995, Takezawa et al. 1996, Poovaiah et al. 1999, Pandey and Sopory 1998) in plants suggests that the above scheme is applicable

to plant cells as well. This review focuses on the Ca2 + -messenger system (step 3) and on some prominent Ca2 + -sensor proteins in plants (step 4); a perspective of Ca2 + -signaling during early plant development viz., embryogenesis, seed development and germination is also discussed.

Ca2 + -messenger system in plant cells As organisms utilize orthophosphate and phosphorylated organic compounds in the cytosolic transduction of free energy, the low solubility product of Ca2 + with Pi would have acted as a driving force for the evolution of mechanisms that maintain low levels of cytosolic Ca2 + . The resulting low cytosolic-Ca2 + levels would have lead to subsequent evolution of Ca2 + -based signaling pathways (Sanders et al. 1999). Research in the past several years has established the critical role of Ca2 + as a second messenger in the coupling of an array of external signals to specific responses (Table 1). Although molecules such as H + (pH), lipids, IP3, cAMP, cGMP, nitric oxide, and cyclic ADP-ribose also act as second messengers in plants, extensive cross-talk occurs between these messengers and the major Ca2 + -mediated signaling cascades in plant cells (Sanders et al. 1999, Kurosaki 1997, Volotovski et al. 1998, Jin and Wu 1999, Durner et al. 1998, Durner and Klessig 1999, Bauer et al. 1999, Jacob et al. 1999, Reddy 2001). In addition, plants have the ability to increase the cellular content of Ca2 + -mediated signal intermediates as a response to diverse stimuli (Table 2). The involvement of Ca2 + in signal/response coupling can be experimentally determined by demonstrating stimulusinduced elevation in [Ca2 + ]cyt. However, it is necessary to also show that blocking of such an elevation would arrest the

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Table 2. Stimulus-induced increase in Ca2 + -mediated signaling intermediates. Stimulus

Increase in cellular content

Reference

Rain, wind and touch Wounding Venom peptides mellitin and mastaporan Dehydration and salt stress Abscisic acid Various

CaM and CaM related gene expression IP3 Phospholipase C cADP-ribose CDPK

Braam and Davis (1990) Bergey and Ryan (1999) Drobak and Watkins (1994) Hirayama et al. (1995) Wu et al. (1997) See Table 10

downstream response. This can be demonstrated by challenging the cells with Ca2 + -channel blockers and Ca2 + -chelators. Furthermore, it can be shown that an artificially induced elevation of [Ca2 + ]cyt can result in downstream response even in the absence of the primary stimulus. In addition, plantCa2 + -sensor proteins necessary to transduce the signal further downstream must be detected, isolated, and characterized.

Measurement of cytosolic Ca2 + Ca2 + measurements were first made possible by using Ca2 + selective microelectrodes and the single wavelength fluorescence-Ca2 + -indicator quin-2 (Tsien 1980, 1983, Tsien et al. 1982). Quin-2 has major limitations, such as short excitation wavelength, low extinction coefficient, low fluorescence quantum yield, and a high affinity for Ca2 + (Tsien 1980, Tsien et al. 1982). Efforts to overcome such limitations with Ca2 + sensitive dyes led to the development of a new generation Ca2 + indicators, such as indo-1 and fura-2 (Grynkiewicz et al. 1985). Both these dyes exhibit stronger fluorescence, weaker affinity for Ca2 + , and exhibit shifts in fluorescence spectra upon Ca2 + -binding, and therefore are ratiometrically analyzed. Although advances have been made with respect to fluorescent Ca2 + -dyes, both microinjection and passive loading of these probes into plant cells are associated with extensive technical difficulties. To circumvent this, a novel approach was developed that uses transgenic plants expressing the photoprotein apoaequorin for plant intracellular Ca2 + measurements (Knight et al. 1991). Aequorin is ideal for Ca2 + measurements as it is nontoxic, non-perturbing, has no problems of leakage and compartmentalization, does not require an excitation light, is a very weak buffer at physiological pCa values (Miller et al. 1994), and has a 1000-fold effective dynamic range, from 0.1 to 100 µmol/L (Blinks 1989). Aequorin has been widely used in monitoring cytosolic Ca2 + -influxes in plants induced by various stimuli, such as heat shock (Malho et al. 1998), hypo- and hyperosmotic shock (Trewavas 1999, Takahashi et al. 1997), elicitor, blue light, cold shock, anoxia, oxidative shock (Trewavas 1999, Reddy 2001), etc. As advancement over this method, cell-type- (Wood et al. 2000, Kiegle et al. 2000 a) and organelle/intracellular location-spec-

ific (van der Luit et al. 1999, Knight et al. 1996, Pauly et al. 2000, Reddy 2001) targeting of aequorin have also been carried out in certain transgenic lines. Nonetheless, aequorin has limitations, such as difficulties in imaging its luminescence since it emits less photons than fluorescence, and the necessity of incorporating the cofactor coelenterazine into cells. Such limitations are overcome with the recently developed Ca2 + indicators, ‹cameleons›, since they combine the advantages of protein targeting and fluorescent Ca2 + probes (Miyawaki et al. 1999). Cameleons are chimeric proteins consisting of green fluorescent protein variants and CaM and can be targeted to specific intracellular sites to monitor local Ca2 + dynamics. Yellow cameleon (Miyawaki et al. 1999) has been expressed in Arabidopsis thaliana, and the fluorescence ratio imaging of this indicator was successfully used to measure Ca2 + dynamics in guard cells (Allen et al. 1999 a, b).

Maintenance of ‹resting› [Ca2 + ]cyt in plant cells Plant cells maintain a low resting cytosolic Ca2 + concentration in the approximate range of 10 and 100 nM. However, upon stimulation the cytosol has access to two types of Ca2 + pools/stores. An extracellular pool, viz., the apoplast, and intracellular pool including the vacuole, ER, the mitochondria and possibly the Golgi vesicles. The concentration of Ca2 + in the external and internal pools can be in the range of 0.1 to 10 mmol/L (Table 3). Therefore, under unstimulated conditions, considerable electrochemical potential differences for Ca2 + (∆µCa2 + ) across tonoplast and the plasma membrane are maintained by the energized transport of Ca2 + out of the cytosol. The ∆µCa2 + across the plasma membrane is approximately – 50 kJ mol –1 (Table 3). The potential difference across ER, mitochondria, and plastid membranes has not been determined. Nonetheless, large driving forces probably exist across these endomembranes, making necessary the energized removal of Ca2 + from the cytosol. The transport system that energizes efflux of cytosolic Ca2 + constitutes H + /Ca2 + antiports that use either pmf (proton motive force), ∆pH (Schumaker and Sze 1986), or ∆Ψ (electrical potential) (Blackford et al. 1990), and the calcium ATPases that use ATP hydrolysis to drive Ca2 + up the gradient. The Ca2 + -efflux system thus is responsible for the maintenance of resting

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Veena S. Anil, K. Sankara Rao

Table 3. Distribution of Ca2 + in cellular compartments. [Ca2 + ]cyt

[Ca2 + ]apoplast

[Ca2 + ]organelle

Membrane type

∆µ Ca2 + (kJmol – 1)

∼100 nmol/L1, 2 ∼100 nmol/L ∼100 nmol/L ∼100 nmol/L ∼100 nmol/L

100 µmol/L – 1 mmol/L3, 4 – – – –

– Vacuole 1 mmol/L5, 1 ER 3 – 50 µmol/L6, 7 Plastic-micromolar range8 Mitochondria ∼200 nmol/L1

Plasma membrane Tonoplast ER membrane Plastid membrane Outer mitochondrial

– 45 to – 601 – 28 to – 345, 1 ND ND ND

Key to references: 1 Bush (1995); 2 Sanders et al. (1999); 3 Bjorkman and Cleland (1991); 6 Bush et al. (1989); 7 Bush and Wang (1994); 8 Kreimer et al. (1988); ND = not determined.

[Ca2 + ]cyt and also for the termination of a Ca2 + -signal. In addition, it loads ER, vacuole, and other internal organelles with Ca2 + . These Ca2 + -pools act as sources for stimulusinduced Ca2 + release.

H + /Ca2 + antiporters Antiports are ubiquitous and highly active in plants (Schumaker and Sze 1990). H + /Ca2 + antiports have been reported not only in vacuolar membrane but also in other membranes, including the plasma membrane (Kasai and Muto 1990). Antiporters are inhibited by protonophores and not by vanadate, and thus are easily distinguished from Ca2 + -ATPases. Based on electrogenicity of transport, the stoichiometery of H + exchanged per Ca2 + transported has been proposed to be two (Blumwald and Poole 1986) or three (Blackford et al. 1990). In contrast to the Ca2 + -ATPases, plant H + /Ca2 + antiporters exhibit low affinity for Ca2 + (Km for Ca2 + of 10 – 67 µmol/L) (Bush 1995). However, antiports operate at a higher maximal rate than the Ca2 + -ATPases, and therefore transport comparable amounts of Ca2 + (Bush and Wang 1994). Although plant vacuolar H + /Ca2 + antiport activity was partially purified and reconstituted (Schumaker and Sze 1990), it was only after the characterization of yeast mutants defective in Ca2 + transport that screening and molecular characterization of plant antiporters have become possible. Using this screening method, vacuolar localized Ca2 + /H + antiporters (calcium exchangers) have been identified from Arabidopsis (CAX1 and CAX2) and mung bean and functionally expressed in the mutant yeast (Hirschi et al. 1996, Ueoka-Nakanishi et al. 2000). Increased antiport activity caused by expression of CAX1 in transgenic tobacco plants lead to severe depletion of cytosolic Ca2 + levels, resulting in abnormalities (Hirschi 1999).

P-type Ca2 + -ATPases The Ca2 + -ATPases belong to an evolutionarily diverse group of single, subunit primary ion pumps that form phosphorylated intermediates (phospho-aspartate) during transport,

4

Harker and Venis (1991);

5

Felle (1988);

and are hence called P-type ATPases. In non-plant organisms, the subtype of P-type ATPases located at the ER membrane is termed ER-type or type IIA (Schatzmann 1989) and the subtype located at the plasma membrane is termed PMtype or type IIB pumps (Bush et al. 1989) (Table 4). P-type Ca2 + -ATPase is ubiquitous in plants and has been detected in all types of membrane preparations (Dupont et al. 1990, Graf and Weiler 1989, Rasi-Caldogno et al. 1989, Bush and Sze 1986, Brairs et al. 1988, Bush et al. 1989). Like its animal counterpart, it is inhibited by vanadate, forms phosphorylated intermediates, has high affinity for Ca2 + (Rasi-Caldogno et al. 1989, Liss et al. 1991) and low affinity for ATP (Chen et al. 1993, Williams et al. 1990). In addition, biochemical and molecular evidence suggests that the P-type Ca2 + -ATPases of plants can also be subdivided into the type IIA and type IIB pumps. Although plant-type IIB pumps are CaM-stimulated and share many properties with the PM-type ATPases in animal cells, they are located in the ER and tonoplast and very rarely at the plasma membrane (Bush 1995) (Table 4). They also differ from the animal type IIB pumps in having an autoinhibitory domain at the N-terminal end and not at the C-terminal region of the polypeptide (Harper et al. 1998). Several genes encoding type-IIA pumps have also been cloned from plants (Sanders et al. 1999). However, unlike the animal prototype, these pumps have been localized to non-ER locations like the tonoplast and the plasma membrane. The typical stoichiometry for animal-type-IIA pumps of 2 Ca2 + transported per ATP hydrolyzed seems unlikely for plant type-IIA pumps at the plasma membrane due to the large ∆µCa2 + . In addition, there is evidence to suggest that plant-type IIA pumps at the plasma membrane may function as antiporters, moving two protons in the opposite direction for every Ca2 + transported (Felle et al. 1992, Miller et al. 1990, Rasi-Caldogno et al. 1989). The plant type-IIA pump, similar to its animal counterpart, is not CaM-stimulated and it does not bind CaM (Liang and Sze 1998). The regulatory mechanism of the plant-type-IIA pumps remains unclear, as unlike the animal-type-IIA pumps, they lack the binding motif for exogenous inhibitor protein, phospholamban (Toyofuku et al. 1994, Nakamura et al. 1998) (Table 4). Phospholamban itself has not been detected so far in plants (Geisler et al. 2000).

Ca2 + -mediated signaling in plants

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Table 4. A comparison of non-plant and plant-type IIA and IIB Ca2 + -ATPases. Sub-type

Mr (kD)

CaM-↑

AcidicPhospholipid ↑

Formation of Phosphorylated intermediates by La3 + -↑↓

Non-Plant organisms Type IIA1, 2, 6 100 – 120 134 – 140 Type IIB1, 3

– +

– +

↓ ↑

Plants Type IIA1, 4 Type IIB1, 4, 5

– +

– +

ND ↑

100 – 120 100 – 140

Key to references: 1 Geisler et al. (2000); 2 Schatzmann (1989); al. (1991); ND = not determined; ↓ = inhibition; ↑ = stimulation.

3

Signal-induced Ca2 + homeostasis Electrophysiological and biochemical studies strongly suggest the existence of voltage-gated, stretch-activated, and ligand-activated Ca2 + -channels in plants (Table 5) that are responsible for signal-induced cytosolic-Ca2 + -elavations. Voltage-gating is critical in channel activation, as propagation of many signals such as blue light (Spalding and Cosgrove 1989), nodulation factors (Ehrhardt et al. 1992), fungal elicitors (Kuchitsu et al. 1993), red light (Ermolayeva et al. 1996), and cell elongation (Kiegle et al. 2000 b) involve rapid membrane depolarization or hyperpolarization in plants. Recently, hyperpolarization-activated Ca2 + channels have been implicated during the ABA-induced increases in cytosolic Ca2 + in guard cells (Hamilton et al. 2000) and during tip growth of Arabidopsis root hairs (Very and Davis 2000). Some hyperpolarization-activated Ca2 + -channels, termed ‹mechano-sensitive›, are stretch-activated (Table 5) and are proposed to transduce mechanical stress induced by turgor, gravity, touch, or flexure (Pickard and Ding 1993). Slow-activating vacuolar (SV) channels are depolarization-activated Ca2 + -channels that are stimulated at [Ca2 + ]cyt over 600 nM (Hedrich and Neher 1987), and are implicated in Ca2 + -induced Ca2 + release (CICR) in plant cells (Sanders et al. 1999). Prominent among the ligand-gated Ca2 + channels is the IP3-activated Ca2 + channel found both at the endomembranes and at the plasma membrane of plant cells (Poovaiah and Reddy 1993, Sanders et al. 1999). The involvement of IP3gated channels has been suggested in osmo-regulation (Srivatsava et al. 1989, Cho et al. 1993), stomatal closure (Gilroy et al. 1990), red light-induced modulation of turgor in the motor cells of leaf pulvini (Kim et al. 1996), and in self-incompatibility and reorientation responses of pollen tubes (FranklinTong 1999). Furthermore, some Ca2 + -channels located at the vacuolar membrane are cyclic ADP-ribose (cADPR)-activated and show homology to ryanodine receptors (Muir and Sanders 1996). Microinjection of both IP3 and cADPR into guard cells has shown that both these second messengers have the

Location

(Auto)inhibitor

ER-membrane Plasma membrane

Exogenous protein (Phospholamban) Endogenous (C-terminal)

Plasma membrane ER-membrane tonoplast

ND Endogenous (N-terminal)

Bush et al. (1989);

4

Bush (1995);

5

Sanders et al. (1999);

6

Chiesi et

capacity to elevate [Ca2 + ]cyt, thereby demonstrating that IP3 and cADPR-gated channels are functional in Ca2 + release in plant cells (Gilroy et al. 1990). In addition, a Ca2 + -channel detected at the plasma membrane of parsley suspension cells, termed ‹large-conductance elicitor-activated channel› (LEAC) (Table 5), is fungal elicitor-activated and involved in the induction of defense-related gene expression. More recently, Schroeder’s group has identified a novel plasma membrane Ca2 + -channel that is activated by H2O2 and is involved in ABA signaling in guard cells (Pei et al. 2000).

Decoding the calcium message Plants use a single messenger system in the coupling of diverse signals with a wide array of physiological responses. Different environmental and hormonal signals can specifically mobilize Ca2 + into the cytosol, nucleus, or chloroplast from different Ca2 + -pools, such as the cell wall, ER, vacuole or mitochondria (Sanders et al. 1999, Reddy 2001). More importantly, different stimuli can generate cytosolic-Ca2 + elevations that vary enormously in amplitude, kinetics, and spatial distribution (Trevawas 1999, Bush 1995). Signals can stimulate Ca2 + -influx either as a transient spike (Knight et al. 1996, Takahashi et al. 1997, Volotovski et al. 1998, Anil and Sankara Rao 2000), as Ca2 + oscillations (Trewavas and Malho 1997), or as a wave. Well-studied examples for Ca2 + -waves are the tip-focused Ca2 + gradients in pollen tubes (Rathore et al. 1991, Miller et al. 1992, Franklin-Tong et al. 1997), root hairs (Herman and Felle 1995), and algal rhizoids (Brownlee and Wood 1986, Taylor et al. 1996). Such a calcium wave can be artificially induced by the photolysis of caged IP3 or caged Ca2 + (Franklin-Tong et al. 1996, Malho and Trewavas 1996). This wave initiates in the proximity of the nucleus and ER and moves to the tip within one minute via a relay of IP3-sensitive channels. The Ca2 + -channel activity is higher at the pollen tube tip, thus resulting in higher concentrations of Ca2 + in this region (Malho et al. 1995). Therefore, the shape or topology of the calcium wave depends on the location and manner in

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Veena S. Anil, K. Sankara Rao

Table 5. Ca2 + -channels in plant cells. Type

Name

Tissue

Location

Inhibitors of Ca2 + flux

Depolarizationactivated

(1) DACC1, 2 (2) DACC3 (3) rea4, 5

carrot cell suspension Arabidopsis root Wheat root

plasma membrane plasma membrane plasma membrane

Rye root Rye root Various Tobacco suspension

plasma membrane plasma membrane tonoplast tonoplast

ND Mibefradil Ruthenium red, verapamil, La3 + , Al3 + , Ni2 + , Gd3 + , diltiazem Verapamil, La3 + Ruthenium red Amiloride Cd2 +

Vicia faba guard cell Onion bulb epidermis, Arabidopsis root and leaf Beet root Beet root Arabidopsis root hairs Vicia guard cells Arabidopsis roots

plasma membrane plasma membrane

ND Al3 + , La3 +

tonoplast tonoplast plasma membrane plasma membrane plasma membrane

Nifedipine, verapamil Zn2 + , Gd3 + La3 + , Gd3 + , Al3 + [Ca2 + ]cyt Verapamil, Co2 + , Al3 +

Parsley cell suspension Beet root Beet root Vicia faba guard cell Arabidopsis guard cells

plasma membrane tonoplast tonoplast tonoplast plasma membrane

La3 + , Gd3 + Verapamil, heparin, TMB8 Ruthenium red, procaine [Ca2 + ]cyt ND

(4) (5) (6) (7) Hyperpolarizationactivated

(1) Mechanosensitive10 (2) Mechanosensitive11 (3) (4) (5) (6) (7)

Ligand-activated

VDCC24 Maxi-cation4 SV6, 7, 8, 17 Depac 29

Hypac 112 Hypac 213, 17 NN18 NN19 NN20

(1) LEAC14 (2) IP3-dependent15 (3) cADPR-dependent16 (4) H2O2-activated21

DACC = Depolarization-activated Ca2 + -permeable channel; SV = slow vacuolar channel; LEAC = large-conductance elicitor-activated channel; VDCC = voltage-dep. Ca2 + channel. Key to references: 1 Thuleau et al. (1994 a); 2 Thuleau et al. (1994 b); 3 Kiegle et al. (1998); 4 White (1998); 5 Pineros and Tester (1997); 6 Schulz-Lessdorf and Hedrich (1995); 7 Hedrich and Kurkdjian (1988); 8 Weiser and Bentrup (1993); 9 Ping et al. (1992); 10 Cosgrove and Hedrich (1991); 11 Pickard and Ding (1993); 12 Gelli and Blumwald (1993); 13 Johannes et al. (1992); 14 Zimmermann et al. (1997); 15 Alexandre and Lassalles (1992); 16 Leckie et al. (1998); 17 White (2000); 18 Very and Davies (2000); 19 Hamilton et al. (2000); 20 Kiegle et al. (2000 b); 21 Pei et al. (2000); ND = not determined; NN = not named.

which channels are located in relation to one another at the membranes. The stimulus-specific spatial distribution and unique kinetics of a Ca2 + influx seem to dictate the activation of unique combinations of downstream calcium-sensor proteins resulting in specific response. Moreover, the same Ca2 + signal in different cell types can initiate different downstream responses, adding further to the complexity of the Ca2 + -code in plants.

signaling pathways. A number of Ca2 + -binding proteins have been identified and implicated in a wide spectrum of physiological functions in plants (Table 6). Most of these Ca2 + binding proteins have the EF-hand motif similar to that found in CaM. This review emphasizes CaM and the unique CaMlike domain protein kinase (CDPK) since they are implicated in the regulation of a wide range of cellular functions in plants.

Calmodulin

[Ca2 + ]cyt sensors and the activation of protein kinases Plant proteins that sense changes in [Ca2 + ]cyt Ca2 + has the ability to coordinate six to eight uncharged oxygen atoms that allow remote domains to participate in Ca2 + binding and bring about conformational changes in proteins (McPhalen et al. 1991). Many organisms, including plants, have evolved mechanisms to exploit Ca2 + -binding-induced conformational changes to activate downstream events in

Detailed structural analysis of mammalian calmodulin (CaM) reveals that it is a small molecular weight (∼14–19 kD), dumbbell-shaped protein having two Ca2 + -binding sites (EF-hands) per lobe (Babu et al. 1988). An EF hand constitutes an N-terminal helix (E-helix), a Ca2 + co-ordinating loop comprising 12 amino acids, followed by a C-terminal helix (F helix) (Kretsinger 1976, Klee and Vanaman 1982). The crystal structure of mammalian CaM (Babu et al. 1988) reveals that each of the Ca2 + -binding loops exhibits a seven-fold co-ordination with Ca2 + (Fig. 1). CaM regulates the activity of a number of downstream CaM-binding proteins (CBPs). Recent studies show

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Table 6. Calcium-binding proteins in plants. Ca2 + -binding protein

Source

CaM

Carrot somatic embryos Anthers of lily Ubiquitous Wheat cultured cells Lotus japonicus: root and nodules Rice suspension culture cells Brassica pollen (allergens) Birch pollen (allergens) Tobacco Pisum sativum: vegetative and flowering tissues Oat seedlings Plant oil bodies Bean

CCD-1 (ccd-1-encoded protein) LjCbp1 (Lotus japonicus Ca2 + -binding protein) CRO1 (rice calreticulin) Bra r 1 and Bra r 2 Bet v 4 BY-2 centrins Calnexin

Caleosin Hra32 (hypersensitive reaction associated) BPC1 and APC1 (B. napus and Arabidopsis pollen calcium-binding protein 1) PCP SOS3 (homologous to calcineurin B of Yeast and animals) Annexins (p33 and p35) CCaMK CDPKs

No. of EF-hands

Reference

1 4

Cell division at apical region Androgenesis Regulation of type IIB Ca2 + -ATPases Defense against fungi Symbiosis between legumes and rhizobia

1 2 3 4 5

– 2 2 4 –

Regulation of regeneration Pollen-pistil interaction ND Formation of cell plate during cytokinesis ND

6 7 8 9 10

1 4

Molecular chaperones in assembly of V1V0-ATPases ND Component of hypersensitive cell death

11 12 13

Arabidopsis and B. napus pollen

2

Pollination

14

Brassica: Pistil and anthers Arabidopsis thaliana

– –

Pollen-pistil interaction, pistil development Salt tolerance

15 16

– 3 (Visinin-like domain) 4

Growth inhibition during mechanical stress Anther development

17 18

Various

19

Bryonia dioica internodes Lilium anthers Ubiquitous

4

Probable physiological role

Key to references: 1 Overvoorde and Grimes (1994); 2 Poovaiah et al. (1999); 3 Bush (1995); 4 Takezawa (2000); 5 Webb et al. (2000); 6 Li and Komatsu (2000); 7 Okada et al. (1999); 8 Grote et al. (1999); 9 Stoppin-Mellet et al. (1999); 10 Ehtesham et al. (1999); 11 Li et al. (1998 b); 12 Chen et al. (1999); 13 Jakobek et al. (1999); 14 Rozwadowski et al. (1999); 15 Furuyama and Dzelzkalns (1999); 16 Liu and Zhu (1998); 17 Thonat et al. (1997); 18 Patil et al. (1995); 19 see Table 10.

Figure 1. Amino acid sequence that constitutes the Ca2 + binding loop of each of the four EF hands of CaM. The residues at positions 1, 3, 5 and 7 (residues in gray), each contributes an oxygen that co-ordinates Ca2 + . The 12th residue in each loop is a glutamate, which binds Ca 2+ through both its carboxylate oxygens. A water molecule completes the seven-fold co-ordination shell. The water molecule is hydrogenbonded to the side-chain of aspartate at 3rd position and also the side chain of amino acids in position 9 of loops 2, 3 and 4 (underlined residues).

that Ca2 + -binding induces conformational changes that expose two hydrophobic clefts in CaM (Babu et al. 1988, Zhang et al. 1995). These clefts interact with aromatic or long aliphatic side chains of the target proteins leading to biological re-

sponse (Chin and Means 2000, Zhang et al. 1995, O’Neil and DeGrado 1990, Gellman 1991). CaM is ubiquitous in eukaryotes, and its presence in plants was reported more than two decades ago (Anderson et al. 1980, Muto and Miyachi 1977). With their animal counterpart, plant CaMs show strong sequence homology (Marme 1988), and similarity in amino acid composition (Klee and Vanaman 1982, Marme 1988) and biochemical properties. Furthermore, spectral analysis suggests that both plant and animal CaM have similar secondary structures and undergo similar conformational transitions upon Ca2 + -binding (Klee and Vanaman 1982, Marme 1988, Dieter et al. 1985, Chin and Means 2000). Plant CaM has been implicated in the regulation of diverse cellular functions. For instance, CaM is involved in the regulation of Rubisco assembly in the chloroplast via the CaM binding Chaperonin 10 (CPN10) (Yang and Poovaiah 2000 a). Recently, Yang and Poovaiah (2000 b) provided evidence for the involvement of Ca2 + /CaM-mediated signaling in the ethylene-regulated senescence and death of leaves and petals.

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Veena S. Anil, K. Sankara Rao

Figure 2. Domain structure of related Ca2 + stimulated protein kinases. CCaM kinase and CDPK can directly bind Ca2 + by means of the endogenous visinin-like/CaM-like domains. Black box represents a Ca2 + -binding site or EF-hand. The N-terminal domain has variable length and sequence.

CaM has been shown to be involved in the inhibition of an actin-bundling protein (P-135-ABP), thus regulating the organization of actin filaments at the pollen tube tip (Yokota et al. 2000). CaM negatively regulates the unique plant microtubule motor protein (KCBP) which is differentially involved in cell division (Vos et al. 2000). Moreover, cyclic nucleotide-gated ion channels from Arabidopsis thaliana have the ability to bind CaM, suggesting that these channels are regulated by CaCaM (Kohler and Neuhaus 2000). CaM is also implicated in sugar-induced anthocyanin biosynthesis in Vitis vinifera cells (Vitrac et al. 2000). In addition, as in animals, plant CaM is involved in the regulation of enzymes, such as calcinurin, NAD kinase, Ca2 + -ATPase, and nuclear NTPases (Poovaiah and Reddy 1993, Bush 1995, Marme 1988, Bonza et al. 2000, Chung et al. 2000, Hwang et al. 2000). In a surprising deviation from animals (Hanson and Schulman 1992, Anderson and Kane 1998, Kane and Means 2000), plants do not use CaM-dependent protein kinases for multifunctional signalintegration. Several initial studies indicated the presence of CaM-dependent protein kinase activity in crude plant extracts (Veluthambi and Poovaiah 1984 a, 1984 b, Blowers and Trewavas 1989, Ranjeva and Boudet 1987). However, it was only recently that Poovaiah and his group were successful in isolating and characterizing a Ca2 + /CaM-dependent protein kinase (CCaMK) from lily, thus providing molecular and biochemical evidence for the presence of a CaM kinase-like enzyme in plants (Patil et al. 1995, Takezawa et al. 1996). Unlike mammalian CaM kinases, the lily Ca2 + /CaM-dependent protein kinase has a neural visinin-like Ca2 + -binding domain in addition to a CaM-binding domain (Fig. 2), and requires binding of both Ca-CaM and Ca2 + for optimal activation (Takezawa et al. 1996). CCaMK has been detected in only a few plant species, such as lily, tobacco, and maize (Patil et al. 1995, Takezawa et al. 1996, Poovaiah et al. 1999, Pandey and Sopory 1998), thus, its ubiquity in plants is yet to be established. Furthermore, the lily and tobacco CCaMK are specific to anthers, suggesting a restricted physiological role. There is also a single report for a more typical CaM kinase in plants (Watillon et al. 1995) that has a CaM-binding site, but lacks the visininlike Ca2 + -binding domain (Fig. 2).

Ca2 + -dependent protein kinase or CaM-like domain protein kinase ‹Calcium-dependent› or ‹CaM-like domain› protein kinases (CDPKs) are responsible for the predominant Ca2 + -dependent, CaM independent protein phosphorylation in plants (Harmon et al. 1986, Putnam-Evans et al. 1990, Roberts and Harmon 1992, Roberts 1993). CDPKs are found only in plants and in a few protozoans, such as Paramecium (Gunderson and Nelson 1987) and Plasmodium falciparum (Zhao et al. 1994). CDPKs are independent of CaM since their N-terminal catalytic domain is fused, via a junction domain, to a C-terminal CaM-like regulatory domain (Fig. 2) (Harper et al. 1991, Urao et al. 1994, Harbak et al. 1996, Hong et al. 1996, Kawasaki et al. 1993, Estruch et al. 1994, Botella et al. 1996). The predicted amino acid sequence of the first-cloned CDPK from soybean (CDPKα) (Harper et al. 1991) shows strong homology to nucleotide-binding site and other regions characteristic of the catalytic domain of Ser/Thr protein kinases between residues 41-328 (Fig. 3). The CaM-like regulatory domain of the prototype CDPKα shows 39 % amino acid identity to spinach CaM and stretches from residue 329-473. It constitutes four putative Ca2 + -binding sites characteristic of helix-loophelix or EF-hand Ca2 + -binding protein family (Fig. 2). Mutational and biochemical analysis of recombinant CDPKs (Harper et al. 1994, Harmon et al. 1994) suggests that in the absence of Ca2 + , the junction domain acts as a pseudosubstrateautoinhibitor, and binds to the catalytic domain, keeping it intrinsically inactive. According to the model for activation of CDPK (Huang et al. 1996), Ca2 + -binding induces the CaMlike domain to disengage the autoinhibitor from the kinase domain, thus activating the enzyme (Fig. 4). Corroboratively, CD analysis of Plasmodium falciparum CDPK (PfCPK) (Zhao et al. 1994) and intrinsic fluorescence properties of swCDPK (Anil 2000, Anil and Sankara Rao 2001) are suggestive of Ca2 + -binding-induced conformational changes in the enzyme. It has been demonstrated that different substrates can interact with CDPK to bring about changes in the enzyme’s Ca2 + binding properties (Lee et al. 1998). Furthermore, some isoforms, in addition to Ca2 + , require phospholipids (Binder et al.

Ca2 + -mediated signaling in plants

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Figure 3. Alignment of predicted amino acid sequence of soybean CDPK with CaM kinase II and CaM. Identical residues are indicated by *. The CDPK sequence is divided at the boundary between the kinase and CaM-like domains. Boxes a, b, and c are diagnostic sequences and boxes I, II, III and IV are calciumbinding sites. Taken from Harper et al. (1991).

Figure 4. CDPK activation model as proposed by Huang et al. 1996. The kinase domain (K) is kept intrinsically inactive by the binding of the endogenous autoinhibitory-junction (J). In presence of Ca2 + , the CaM-like domain (CaM-LD) binds J, disengages it from K, thus activating the enzyme.

1994, Farmer and Choi 1999, Szczegielniak et al. 2000) or 143-3 proteins (Camoni et al. 1998) for maximal activation. All CDPKs, except isoforms from winged bean (wbCDPK, wbCPK), exhibit intramolecular Ca2 + -dependent autophosphorylation (Table 7). Experimental evidence suggests that autophosphoryation does not change CDPK’s requirement of activators (Chaudhuri et al. 1999, Harmon et al. 2000), nor does it prevent the intrinsic inhibition by the autoinhibitor (Harmon et al. 1994, 2000). Nevertheless, it has been demonstrated with CDPKs from both alfa-alfa (Borge et al. 1988) and groundnut (Chaudhuri et al. 1999) that autophosphorylation increases rates of substrate phosphorylation activity, indicating an up-regulatory role for this activity. In contrast, Saha and

Singh (1995) demonstrated that autophosphorylation of wbCDPK results in reduced substrate phosphorylation. Moreover, neither up- nor down-regulatory effects of autophosphorylation have been detected in the prototype soybean CDPK under comparable assay conditions (Roberts and Harmon 1992). Such divergent observations suggest that any regulatory function of autophosphorylation cannot be generalized for CDPKs, and the regulatory or physiological significance of this activity remains unclear. CDPKs are ubiquitous in plants, and several of their features give them credibility as key intermediates in Ca2 + mediated signal transduction. The most prominent of such features is the ability to be stimulated by submicromolar to micromolar levels of Ca2 + in vitro. These levels of Ca2 + fall in the range of stimulus-induced elevation of [Ca2 + ]cyt in plant cells. In addition, as is clear from Table 9, both activity and expression of CDPKs can be induced by various environmental and hormonal stimuli. Moreover, CDPKs exist as multiple isoforms in a single species (Harmon et al. 2000, Harbak et al. 1996), and show tissue-specific and developmentally regulated expression (Table 9) (Hong et al. 1996, Kawasaki et al. 1993, Nishiyama et al. 1999). Variations in enzyme kinetic properties and other biochemical properties (Table 7 and 8) further imply that members of this family of kinases are designed to respond to a multitude of environmental and hormonal stimuli. This presumption is corroborated by several studies that implicate CDPKs in the regulation of a wide range of developmental and cellular functions (Table 10). Furthermore, CDPKs phosphorylate and inhibit a CaM-stimulated Ca2 + -pump (ACA2) (Hwang et al. 2000), and phosphorylate the CaM antagonists, napin large chains (Neumann et al.

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Veena S. Anil, K. Sankara Rao

Table 7. A comparison of the biochemical properties of CDPKs. Autophosphorylation 2+

2+

Ca -dep Ca -indep soybeanCDPK

+

wbCDPK wbPK gnCDPK

– +

+ –

+

potatoCDPK DcCPK CharaCDPK SpinachCDPK1

+ n/d n/d

barleyCDPK maizeCDPK

+

TobaccoCDPK swCDPK from somatic embryos

+ +

Histone III-S

1.7 µmol Ser and Thr 0.07 mg/mL 1.8 nmol n/d 0.035 mg/mL 5.9 nmol – 0.4 µmol Thr 50 µmol/L

Syntide 2 Histone III-S Histone III-S Nitrate reductase (Synthetic peptide) Nitrate reductase (Synthetic peptide) Syntide-2 phosphoenol pyruvate Carboxylase (in vivo) Histone III-S Histone III-S

n/d

SpinachCDPK2

Km

Histone III-S Histone III-S MLCpep

n/d

Vmax min – 1 mg – 1

P-amino acid

substrate used

AP

SP

IC50 ↓↑ S/M W7 SP by µmol/L AP

Mol ReferWt ence kD

0.13 mg/mL

Ser

110

n/d

Soluble

55

1

n/d n/d 30

↓ – ↑

Soluble Soluble Soluble

62 70 53

2 3 4

30 µmol/L n/d n/d 0.8 µmol/L

n/d n/d n/d 60.3 µU

n/d n/d n/d n/d

Ser Ser Ser and Thr n/d n/d n/d n/d

250 66 n/d n/d

n/d n/d n/d n/d

Soluble Soluble Soluble Soluble

53 57 51 45

5 6 7 8

0.6 µmol/L

28.8 µU

n/d

n/d

n/d

n/d

Soluble

60

9

n/d n/d

n/d n/d

n/d n/d

n/d Ser-15

125 +

n/d n/d

Soluble Soluble

54 50

10 11

n/d 1.3 mg/mL

n/d n/d 0.1 nmol Thr

n/d Ser

n/d 6

n/d n/d

membrane 54 Soluble 55

12 13

Key to references: 1 Putnam-Evans et al. (1990); 2 Saha and Singh (1995); 3 Ganguly and Singh (1998); 4 DasGupta (1994), Chaudhuri et al. (1999); 5 MacIntosh et al. (1996); 6 Farmer and Choi (1999); 7 McCurdy and Harmon (1992); 8 Bachmann et al. (1996); 9 Bachmann et al. (1996); 10 Ritchie and Gilroy (1998); 11 Ogawa et al. (1998); 12 Iwata et al. (1998); 13 Anil (2000); n/d = not determined; AP = autophosphorylation; SP = substrate phosphorylation; M = membrane associated; S = soluble; ↓↑ = up and down regulation respectively.

Table 8. A comparison of enzymatic properties of CDPKs. CDPK

Mol. pH

type

Wt

optimum

kD GnCDPK

53

Type

a

ATP

Substrate Km (µmol/L)

Km (µmol/L)

k3 (min – 1)

ATP

Substrate

a

k3/Km (M – 1 min – 1) ATP

Substrate

K05

Reference

Ca2 + (µmol/L)

0.43 × 106 0.43 × 106 4.3 × 1010 8.6 × 109

0.5

Das Gupta (1994)

1.6b

8 6.8

7.5 × 106 3.7 × 103

7.5 × 106 8.3 × 102

1.5 nd

Putnam-Evans et al. (1990) Ganguly and Sing (1998)

Histone III-S

3.3b

16.4

2.5 × 102

1.12 × 102 1.5 × 107

34.1 × 106

nd

Saha and Singh (1995)

Histone III-S

7.8b

3.8

0.12 × 102 0.12 × 102 3.3 × 106

1.6 × 106

nd

Binder et al. (1994)

Histone III-S

61b

0.01

1 × 102

1.6 × 106

0.7

Anil (2000)

9 – 10

MLCpep

Soybean CDPK 55

7–9

Histone III-S

6.1b

50

wbPK

70

nd

Histone III-S

wbCDPK

60

nd

AK-1-6H

97

nd

swCDPK

55

6.8 – 8.5

10

1 × 102

9.3 × 1011 1.2 × 1012 5.4 × 108 5.2 × 108

1 × 1010

n/d = not determined; a Calculated from values obtained from references indicated; b Molar value calculated using 21 kD molecular weight of histone III-S.

1996). These can be considered as examples for potential points of cross-talk between CaM- and CDPK-mediated signal transduction pathways in plants. Nevertheless, the limited information concerning the in vivo substrates and other downstream participants has made it difficult to conclusively elucidate Ca2 + -mediated signaling pathways involving members of this unique family of kinases.

Critical involvement of Ca2 + -signaling in plants Advances in plant cell-, molecular-biology and biochemical techniques have irrevocably established the involvement of Ca2 + -signaling during phytochrome action (Shacklock et al. 1992, Bowler et al. 1994, Neuhaus et al. 1997), stomatal clo-

Ca2 + -mediated signaling in plants

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Table 9. Signal-induced and development-regulated activation/expression of CDPK. CDPK/source plant

Induction of activity

Induction of expression/ development specific expression

Reference

CDPKs from Nicotiana tabacum

–

CDPK from Sorghum CDPK from Maize Vr-CDPK-1 from mung bean

Osmotic stress Osmotic stress

Iwata et al (1998) Yoon et al (1999) Pestenacz and Erdei (1996) Pestenacz and Erdei (1996) Botella et al. (1996)

CDPK from tomato CDPK from Rice CDPK from Potato OsCDPK2 from rice

Fungal elicitor – Tuber development –

OsCDPK11 from rice CDPK from Funaria hygrometrica CDPK from maize swCDPK from sandalwood endosperm swCDPK from sandalwood zygotic embryo

– Indole-acetic acid, pH 5 and nitrate starvation – –

Sucrose, phytohormones, methyl jasmonate, wounding, fungal elicitors, chitosan and NaCl – – CaCl2, salt stress, indole-3-acetic acid, mechanical strain – gibberellin – Darkness, flower development and later stages of seed development Early seed development

Late stages of pollen development Seed maturation and germination

Estruch et al. (1994) Anil et al. (2000)

Embryogenesis, dormancy and germination

Anil et al. (2000)

Cold and salt/drought stress

Martin and Busconi (2001), Saijo et al. (2000)

CDPKs from rice

Embryogenesis and germination (inactive during dormancy) Cold

Romeis et al. (2000) Abo-el-Saad and Wu (1995) MacIntosh et al. (1996) Frattini et al. (1999) Frattini et al. (1999) D’Souza and Johri (1999)

Table 10. Diverse physiological functions of CDPKs. CDPK/source plant

Implicated physiological role

Reference

CDPK from potato CDPK from Chara OsCDPK11 from rice OsCDPK2 from rice CDPK from Funaria hygrometrica CDPK from barley aleurone CDPK from maize CDPKs from spinach CDPK from maize and soybean

Tuber development Cytoplasmic streaming Seed development Seed development and response to light Differentiation of caulonema Gibberellin-response during germination Regulatory phosphorylation of phosphoenolpyruvate carboxylase Regulatory phosphorylation of nitrate reductase Regulatory phosphorylation of sucrose synthase

CDPK from spinach VrCDPK from mung bean CDPK from maize and Sorghum CDPK1 and CDPK1a CPK1 CDPKs from rice

Regulatory phosphorylation of sucrose phosphate synthase Salt- and mechanical-stress response Drought-stress response Positive regulators of stress signal transduction Inhibits a CaM-stimulated Ca2 + pump (ACA2) located at ER Regeneration of rice cultured cells Lamina inclination Defense response against fungal-pathogen Cladosporium fulvum Pollen germination and pollen tube growth Mobilization of nutrient stores during germination Embryogenesis and germination Salinity and drought stress response Regulatory phosphorylation of K + channel in guard cells

MacIntosh et al. (1996) McCurdy and Harmon (1992) Frattini et al. (1999) Frattini et al. (1999) D’Souza and Johri (1999) Ritchie and Gilroy (1998) Ogawa et al. (1998) Bachmann et al. (1996) Huber et al. (1996); Zhang and Chollet (1997) Mc Michael et al. (1995) Botella et al. (1996) Pestenacz and Erdei (1996) Sheen (1996) Hwang et al. (2000) Karibe and Komatsu (1998) Yang and Komatsu (2000) Romeis et al. (2000) Estruch et al. (1994) Anil et al. (2000) Anil et al. (2000) Patharkar and Cushman (2000) Li et al. (1998 a)

CDPK from tomato CDPK from maize swCDPK from sandalwood endosperm swCDPK from sandalwood embryos Mc CDPK1 CDPK from Vicia faba

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Veena S. Anil, K. Sankara Rao

sure (MacRobbie 1997, Allen et al. 1999 b, Pei et al. 2000) and tip growth of root hairs, algal rhizoids and pollen tube (Brownlee and Wood 1986, Wymer et al. 1997, Holdaway-Clarke et al. 1998, Malho et al. 1998). Ca2 + -signaling is also involved during diverse responses such as maintenance of circadian rhythm, growth adaptations, root hair curling, and various stress-responses (see Table 1). In addition, research into plant – pathogen interaction (Blumwald et al. 1998) provides strong evidence for the pivotal role of Ca2 + during pathogen defense response (Dietrich et al. 1990, Yang et al. 1997). Perception of microbial pathogens occurs by the interaction of pathogen-derived signals (elicitors) with specific receptors residing in the plant plasma membrane (Nurnberger 1999). Although the understanding of signal transduction cascades that lead to defense response is fragmentary, it is clear that initial receptor – ligand interaction activates specific Ca2 + -channels, elevates cytosolic Ca2 + (Zimmermann et al. 1997, Blume et al. 2000, Grant et al. 2000), activates transcription of CDPK gene (Murillo 2001), induces specific protein phosphorylations (Dietrich et al. 1990, Lecourieux-Ouaked et al. 2000), and elevates reactive oxygen species (Lamb and Dixon 1997). However, an in-depth discussion of the current understanding of these signaling pathways in plants is beyond the scope of this review. Here our main aim is to highlight the current status of research on signaling events controlling early development in plants.

Ca2 + -signaling during early plant development Seed development Seed development involves two vital processes, viz., endosperm development and embryogenesis. Although auxins are produced in pollen, endosperm, and in the embryo of developing seeds, a regulatory role for this hormone during embryogenesis has not yet been elucidated. The small size and relative inaccessibility of embryos in the seeds has contributed to the lack of information on the signaling events occurring during zygotic embryogenesis. Nonetheless, the ability to induce plant embryogenesis from somatic cells has made possible the study of likely intermediates of signaling cascades that regulate embryo development. The auxin 2,4-dichlorophenoxyacetic acid (2,4-D) brings about dedifferentiation from explants of various plant species, and also induces embryogenic competence in the callus. However, subsequent stimulation for embryogenesis occurs by auxin withdrawal. Removal of 2,4-D from the medium induces embryo differentiation from proembryogenic cell masses (PEMs) in different embryogenic systems (Komamine et al. 1992, Sankara Rao 1996). This in vitro developmental phenomenon can be explained by attributing to 2,4-D a negative regulatory role in the cellular processes necessary for embryogenic devel-

opment. If this hypothesis is true, withdrawal of 2,4-D would result in stimulation of the processes leading to cell differentiation and embryo development. Earlier work with carrot somatic embryogenesis indicates that Ca2 + played a greater role than being merely nutritive. Exogenous Ca2 + enhanced embryogenic frequency (Jansen et al. 1990), and its deprivation arrested somatic embryo formation (Overvoorde and Grimes 1994). Ca2 + elevations in the cytosol have not been directly monitored in these studies, yet they suggest an intermediary role for the ion during plant embryogenesis. More recently, using sandalwood somatic embryogenesis system, we have demonstrated a cytosolic Ca2 + spike in proembryogenic cells (PEMs) induced by withdrawal of 2,4-D and increased osmoticum in the medium (Anil and Sankara Rao 2000). These culture conditions are conducive for embryogenic development from PEMs. EGTA in the medium, however, arrested such an elevation in cytosolic Ca2 + , indicating that Ca2 + is mobilized from the exogenous medium. As in the case of carrot somatic embryogenesis, sandalwood embryogenesis is also arrested when grown in differentiation medium containing EGTA or Ca2 + -channel blockers. Nonetheless, these cultures continued to proliferate to form macroscopic cell clumps. Furthermore 35S-metheonine labeling of proteins in these clumps indicates that they exhibit functional protein synthesis machinery. Thus, these observations indicate that chelation of exogenous Ca2 + specifically blocks embryogenesis but does not result in cell death (Anil and Sankara Rao 2000). These observations strongly suggest the second messenger role of exogenous Ca2 + during somatic embryogenesis; whether it is so during zygotic embryogenesis still remains to be determined. Studies with carrot somatic embryogenesis have indicated that the level of CaM mRNA markedly increases in heart- and globular stages of somatic embryogenesis (Overvoorde and Grimes 1994). However, Ca-CaM complexes localized only at the apical meristematic regions of somatic embryos, suggesting that CaM could have a restricted role in these actively dividing cells. Moreover, downstream kinases that are activated by CaM have not been reported from plant embryogenic tissues, minimizing the possibility that signal/response coupling during embryogenesis occurs via the mediation of CaM and CaM-dependent protein kinases. In contrast, Frattini et al. (1999) report temporal patterns of expression of two isoforms of rice CDPK (OsCDPK2 and OsCDPK11) during seed development. While OsCDPK11 is expressed only during early seed development, OsCDPK2 is expressed during flower development and at later stages of seed development. The strong inhibition of early seed development by overexpressing OsCDPK2 (Morello et al. 2000) further suggests diverse physiological roles for these isoforms during seed development. However, whether their physiological role pertains to the endosperm, aleurone, or the embryo is not clear. A recent study has detected swCDPK in mature zygotic embryos and in all stages of somatic embryogenesis (PEMs, globular-, heart-, torpedo- and cotyledonary stages), but not in the

Ca2 + -mediated signaling in plants shoots and flowers (Anil et al. 2000). The accumulation of this early development-specific CDPK in sandalwood embryogenic cultures was blocked when exogenous Ca2 + was chelated from the medium (Anil and Sankara Rao 2000). Furthermore, culture treatment with the CaM/CDPK-inhibitor, W7, arrested embryogenesis (Overvoorde and Grimes 1994, Anil and Sankara Rao 2000), indicating that signaling cascades involving CaM and/or CDPK regulate this developmental process. Another isoform of CDPK (swCPK) was immunolocalized to the storage organelles in sandalwood endosperm cells (Anil et al. 2000). These organelles, now identified as oil bodies, contain neutral storage fat triacyl glycerol (TAG) (Anil 2000). Recent experiments also indicate that oil body association of CDPK is a ubiquitous phenomenon in oil seeds. The detection of in vivo Ser/Thr phosphorylation in the oil body associated proteins during oil accumulation/oil body formation suggests involvement of swCPK during this process (unpublished observations).

Germination The significance of GA during seed germination became clear when it was shown that the embryo synthesizes and releases gibberellins into the endosperm during germination (Lenton et al. 1994). It is now well established that GA promotes the production and secretion of several hydrolytic enzymes, which are involved in the solubilization of the reserves in the endosperm during germination. Nevertheless, the intermediary steps between binding of GA to its cognate transmembrane receptor and the expression of the primary response gene GA-MYB is not completely elucidated. Jones et al. (1998) implicate the heterotrimeric G proteins in GA induction of amylase gene expression in wild oat aleurone layer. Further, barley aleurone protoplasts show a slow rise in cytosolic Ca2 + upon incubation with GA that begins within 1– 4 hours after exposure to the hormone, and precedes the onset of α-amylase synthesis (Bethke et al. 1995, Gilroy and Jones 1992). Wheat aleurone cells also exhibit a steady-state increase in cytosolic Ca2 + a few minutes after treatment with GA (Bush 1996). Abo-el-Saad and Wu (1995) have demonstrated that a rice membrane associated CDPK is highly induced when the seeds are treated for 10 min with GA, and Ritchie and Gilroy (1998) provide evidence that implicates CDPKs in the GA-induced signaling in barley aleurone layer. Therefore, these findings strongly suggest that germination is the end result of signaling pathways that involve Ca2 + and CDPKs. Unlike cereals, dicotyledonous seeds do not possess an aleurone layer. Nevertheless, GA treatment can stimulate germination in dicotyledonous seeds, and mobilization of reserve food in the endosperm or cotyledons occurs directly during germination. Moreover, the increased activity/expression of the early development-specific swCDPK in the embryo during seed germination of Santalum album L. (Anil et al. 2000) indicates the active participation of CDPKs during seed germination in dicots as well.

1249

Perspectives and Conclusions In the last two decades there has been a dramatic unfolding of evidence for the regulatory role of Ca2 + in cellular functions in plants. Ca2 + -mediated signaling in plants is grossly similar to that of animal systems, involving pumps, antiporters, Ca2 + channels, Ca2 + -modulator proteins and protein kinases. Nonetheless, some points at which plants deviate from animal systems are 1) the role of the vacuole as a primary Ca2 + -sink in unstimulated cells and a source for Ca2 + -release in stimulated cells; 2) the ability to increase the cellular concentrations of signaling intermediates when stimulated; 3) the minor significance of plant CaM-kinases in the wide spectrum of Ca2 + -mediated signal/response coupling in plants; and 4) the presence of the unique CaM-like domain protein kinases (CDPKs) and their role as multifunctional signaling integrators. The pivotal role of CDPKs in plant signaling demands that future work include in-depth study of their structure and functional relationship. The major aspects that need to be addressed are 1) the regulatory role of autophosphorylation and its influence on substrate phosphorylation activity; 2) identification of other in vivo activators and inhibitors, and elucidation of their mechanisms of regulation; 3) identification of in vivo substrates which would help elucidate the physiological functions; and 4) understanding Ca2 + -induced conformational changes that would be theoretically essential for the activation of the catalytic domain. Endosperm development, embryogenesis, and germination are dynamic and intriguing developmental processes in the life cycle of plants. However, there are lacunae in our understanding of these processes, and the challenges for the future are many. An increasing number of reports suggest the involvement of Ca2 + -mediated-signaling and CDPKs during germination of cereals and dicot seeds. It is now clear that under conditions conducive for embryo development, exogenous Ca2 + acts as a second messenger and is necessary for somatic embryogenesis (Anil and Sankara Rao 2000). However, though experimental evidence suggests the involvement of CDPK and/or CaM during somatic embryogenesis, we are far from elucidating the signaling cascades that regulate this in vitro development. Even if this pathway were to be elucidated, whether it can be extrapolated to zygotic embryogenesis is again questionable. Demonstrating signal-induced cytosolic Ca2 + elevations in the zygotic embryo is technically difficult and remains a challenge for the future. In summary, plant-signaling cascades are yet to be elucidated completely, and the characterization of components of this system at the biochemical, molecular, and genomic level should be more clearly focused in future research. Acknowledgements. We acknowledge with thanks Prof S. K. Podder for valuable suggestions and critical reading of the manuscript. We also thank Prof C Jayabaskaran for helpful suggestions. Publication of this review is facilitated by grants to the corresponding author from the Council of Scientific and Industrial Research and the Department of Science and Technology, Government of India.

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