D Ca S T P P K: Ecoding 2+ Ignals Hrough Lant Rotein Inases

Annu. Rev. Plant Biol. 2004. 55:26388 doi: 10.1146/annurev.arplant.55.031903.141627 Copyright c 2004 by Annual Reviews.
All rights reserved First published online as a Review in Advance on January 7, 2004
DECODING Ca2+ SIGNALS THROUGH PLANT PROTEIN KINASES

Jeffrey F. Harper,1 Ghislain Breton,1 and Alice Harmon2
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Annu. Rev. Plant Biol. 2004.55:263-288. Downloaded from www.annualreviews.org by University of Uppsala on 12/13/12. For personal use only.
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037; email: harper@scripps.edu, gbreton@scripps.edu 2 Department of Botany, University of Florida, Gainesville, Florida 32611; email: harmon@botany.u.edu
Key Words CDPK, SnRK, CRK, CCaMK, autoinhibition s Abstract Plants harbor four families of kinases that have been implicated in Ca2+ signaling (CDPKs, CRKs, CCaMKs, and SnRK3s). Although each family appears to respond to Ca2+ via different mechanisms, they all utilize Ca2+ sensors that bind Ca2+ through multiple EF-hands. The CDPK (Ca2+-dependent protein kinase) family is represented by the most genes, with 12 subfamilies comprised of 34 isoforms in Arabidopsis and 27 in rice. Some of the calcium-regulated kinases also show potential for regulation by lipid signals and kinase cascades. Thus, Ca2+-regulated kinases provide potential nodes of cross-talk for multiple signaling pathways that integrate Ca2+ signals into all aspects of plant growth and development.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nomenclature: A Very Good Place to Start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subfamilies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SENSOR RESPONDER VERSUS SENSOR RELAY . . . . . . . . . . . . . . . . . . . . . . . . . MODELS FOR Ca2 + REGULATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The CDPK Paradigm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CRK and the Potential for CaM Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CCaMK and Dual Regulation by a Visinin-Like Domain and CaM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SnRK3s and the Signicance of Ca2 + Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . WHY ARE THERE SO MANY DECODERS? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isoform-Specic Ca2 + Activation Thresholds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phospho-Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipid Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subcellular Targeting and Isoform-Specic Complexes . . . . . . . . . . . . . . . . . . . . . . Substrate Specicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COMPARATIVE KIN-OMICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Twelve Subfamilies of CDPKs in Flowering Plants . . . . . . . . . . . . . . . . . . . . . . . . . 1543-5008/04/0602-0263$14.00 264 264 266 267 267 267 269 270 272 272 273 274 275 275 275 276 276
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Regulatory Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Importance of Species-Specic Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression Proling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A REASONABLE WAY TO THINK ABOUT CA2 + -REGULATED KINASES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Does Reasonable Evidence for Ca2 + Regulation Exist? . . . . . . . . . . . . . . . . . . . . . . For Every Ca2+ Signal, CDPKs are Expected to Phosphorylate Many Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONFIRMING REGULATORY NETWORKS IN PLANTA . . . . . . . . . . . . . . . . . . . Phospho-Mimic Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phospho-Site-Specic Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A FUTURE PERSPECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
INTRODUCTION
Ca2+ signals play an important role in many aspects of plant growth and development, including the response of plants to biotic and abiotic stress. In addition, Ca2+ has nutritional functions, including the proper functioning of the secretory pathway and the structural integrity of cell walls. This review focuses on illustrating how a subset of protein kinases may be used to help decode Ca2+ signals in plants. Arabidopsis has 1019 protein kinases (92) (http://plantsp.sdsc.edu), of which 67 have been implicated in Ca2+ signaling. These Ca2+-regulated kinases belong to the CDPK-SnRK (Ca2+-dependent protein kinaseSnf1-related kinase) superfamily (41). The Ca2+-regulated kinases and the principles of Ca2+ signaling in plants have all been well reviewed in recent years (7, 13, 19, 21, 34, 35, 38, 61, 75, 78, 81, 86, 97, 100). The purpose of this review is threefold: First, we review what is known (and not known) about the mechanisms through which Ca2+ regulates different subgroups of plant kinases. Second, we show how a comparison of CDPKs from rice and Arabidopsis can be used to identify potentially important features of different kinase subgroups. Third, we provide a perspective on research directions for the next 5 to 10 years.
Nomenclature: A Very Good Place to Start

The following names have been used to describe members of the CDPK-SnRK superfamily. Figure 1 illustrates the domain structure of each family.
CDPK, Ca2+-DEPENDENT PROTEIN KINASE CPK has been used as the three letter acronym for naming the 34 Arabidopsis CDPKs (sometimes called calmodulindomain protein kinases) (41). These kinases have a unique structure in which a calmodulin-like regulatory domain (CaM-LD) is fused to the C-terminal end of the kinase.
CALCIUM-REGULATED KINASES
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Figure 1 Diagram of kinase structures for members of the CDPK-SnRK superfamily. A shaded box shows the position of the junction sequence in CDPK. The junction contains an autoinhibitor and binding sequence for intramolecular interaction with the CaM-like regulatory domain (CaM-LD). The analogous regions are indicated by shaded boxes (CRK, CCaMK, CaMK) or ovals (SnRK3). The C-terminal regulatory domain of a CDPK is shown with four EF-hands (black). In CRKs, the EF-hands are degenerate (white). In CCaMKs the regulatory domain is most similar to a visinin-like Ca2+-binding protein that contains 3 EF-hands (black). In the remaining kinases there are no EF-hands in the C-terminal domain. Of these, only the SnRK3s have been implicated in Ca2+ regulation. The shaded oval contains a FISL/NAF motif that binds CBLs, which contain 34 EF-hands (see Figure 2). The N-terminal domains are highly variable in length and sequence.
CRK, CDPK-RELATED KINASE CRK has been used to name the eight Arabidopsis protein kinases (41). The name CRK was rst used in 1995 to describe a CRK from carrot in which the CaM-LD had degenerated EF-hands (58). Research on two tobacco isoforms (42, 100) recently provided evidence that some CRKs may be regulated by Ca2+ through their potential interaction with calmodulin (CaM).
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CaMK, CALMODULIN-DEPENDENT PROTEIN KINASE CaMKs are best described in animals systems (e.g., CaMKII and MLCK). They are activated by exogenous CaM binding to an autoinhibitory region located at the C-terminal end of the kinase domain. Arabidopsis does not have a conventional CaMK (41). The best candidate for a conventional CaMK in plants is CB1 from apple (9395).
The name CCaMK was rst used to describe a kinase from lily that harbored a C-terminal regulatory domain more similar to visinin (three EF-hands) than to CaM (68). This feature distinguished it from known CDPKs. Biochemical evidence further suggested that CCaMKs could be activated by exogenous CaM. There is no evidence for CCaMKs in Arabidopsis, but they are present in lily (68), tobacco (59), rice, and the moss Physcomitrella (J.F. Harper & G. Breton, unpublished observation).
SnRK, Snf1-RELATED KINASE The name SnRK was rst proposed for the plant kinases most closely related to Snf1 (32). Snf1 refers to a kinase mutation in yeast that results in sucrose nonfermentation. In Arabidopsis, there are 38 SnRKs divided into three distinct families (41). Some members of the largest family (25 SnRK3s) have been implicated in Ca2+ signaling. This family has also been called CIPK [calcineurin B-like protein interacting protein kinase; (85)] or PKS [protein kinase related to SOS2; (30)].
CCaMK, Ca2+ AND CALMODULIN-ACTIVATED KINASES
CBLs are a family of EF-hand Ca2+-binding proteins (2, 49, 53, 61, 85) that are related to the NCS (neuronal Ca2+sensor) family of proteins (10). They have also been called SCaBPs (SOS3-like Ca2+-binding proteins) (30, 33). In Arabidopsis, 10 CBLs have been identied (30, 61). Their interactions with SnRK3s may regulate kinase activity and subcellular targeting.
CBK, CaM-BINDING PROTEIN KINASE It has been proposed that CCaMKs and CRKs be organized into a group of kinases dened by their ability to bind CaM (100). In some examples, CaM binding has been implicated in activating kinases such as the CCaMKs (88) and some CRKs (42, 100), whereas other kinases display the potential to bind CaM without activation, such as the CRK named OsCBK (99) and the CDPK AtCPK1 (44). We do not use CBK in this review because it does not delineate a functional difference between different members of the CDPKs, CRKs, and CCaMKs.
CBL, CALCINEURIN B-LIKE Ca2+-BINDING PROTEIN
Subfamilies
The hierarchical relationship of kinases used here is superfamily, family, and subfamily, based on sequence similarities. The term group refers to kinases that are grouped together on the basis of a specic feature that may cross family boundaries. For example, the group of CDPKs with potential N-terminal myristoylation
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sites includes members from at least 11 of the 12 CDPK subfamilies. The PlantsP (plants and their phospho-regulatory networks) website (http://plantsp.sdsc.edu/) provides a community resource where issues of nomenclature and functional annotation can be updated, discussed, and cross referenced (90).
SENSOR RESPONDER VERSUS SENSOR RELAY

Although there is evidence for Ca2+ regulation of CDPKs, CRKs, CCaMKs, and SnRK3s, their mechanism of activation can be divided into two different modes. The CDPKs represent sensor responders, in which a covalent connection joins the CaM-like Ca2+ sensor directly to the kinase responder (81). In contrast, the potential regulation of CRKs, CCaMKs, and SnRK3s occurs through an exogenous Ca2+-binding protein (e.g., CBL) or CaM that relays a conformational change to the kinase as a consequence of binding Ca2+. However, a meaningful distinction between a covalently attached sensor and a separate CaBP (Ca2+-binding protein) subunit may become cloudy when one considers that some CaBPs may form stable complexes with their target kinase, which may be functionally equivalent to a covalent attachment under many circumstances. Thus, an interesting question is whether a covalently tethered CaM-LD provides CDPKs with unique functional properties that cannot be replaced by a classic animal-type CaMK. Several speculations can be offered. First, the covalent tethering of a CaM-LD may allow the coevolution of kinases with divergent CaMLDs, thereby generating a set of kinases specialized to decode Ca2+ signals with different magnitudes. A specic Ca2+ sensitivity could then be paired with the phosphorylation of a specic substrate. Second, high rates of cytoplasmic streaming in some plant cells may require a kinase and its Ca2+ sensor to be tightly coupled, thereby avoiding the potential for a nonresponsive kinase due to the dissociation of a diffusible CaM. Third, the complex organization of some plant cells may require an efcient mechanism to cotarget a kinase and its Ca2+ regulator. For example, CDPKs have been found in sieve tubes (98). They are translocated into sieve tubes through plasmodesmatal connections with companion cells. Once in the sieve tube, there is the potential for long-distance transport throughout the plant. In this example, the advantage of a covalently tethered CaM-LD is clear, the kinase and sensor are maintained in a stochiometric relationship and cotargeted to wherever their nal destination may be.
MODELS FOR Ca2+ REGULATION The CDPK Paradigm

Figure 2 shows two alternative models for Ca2+ activation of a CDPK. Model A diagrams a simplied version in which a exible tether joins the CaM-LD to the kinase. Upon Ca2+ stimulation, the CaM-LD binds to the junction and displaces the autoinhibitor. This model is analogous to the activation of an animal CaMK,
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except that the CaM-LD is covalently tethered, unlike CaM, which functions as a separate subunit. Despite the simplicity of Model A, current evidence favors Model B, in which the CaM-LD is prebound to the junction at basal levels of Ca2+. This model was rst proposed to explain (a) mutational evidence that indicated that the tether was not a simple exible linker, but rather provided a structural constraint (91), and (b) circular dichroism studies that suggested that the junction/CaM-LD adopts a bound conformation even at very low Ca2+ concentrations (43). More recent evidence from nuclear magnetic resonance and Ca2+ titration experiments (using AtCPK1) indicates that the CaM-LDs C-terminal lobe adopts a structural conformation that binds Ca2+ with a low nanomolar afnity (Kd), but only when it interacts with the junction domain (J. Christodoulou, J. Harper & W. Chazin, unpublished data). This suggests that under in vivo basal Ca2+ concentrations, the C-terminal lobe is loaded with Ca2+ (open conformation) and is prebound to the junction (as depicted in Figure 2, Model B). This implies that activation occurs through Ca2+ binding to the N-terminal lobe of the CaM-LD. Consistent with this prediction, the N-terminal lobe displayed a Ca2+ Kd around 1 M, as expected for a kinase regulated by typical cytosolic Ca2+ signals. The conformation of the N-terminal lobe presumably changes in response to Ca2+ binding, and somehow induces a conformational change in the autoinhibitor/kinase interaction. Figure 2 diagrams working models for CRKs, CCaMKs, and SnRK3s. These diagrams contain question marks to emphasize their speculative nature. The models are presented from the perspective of CDPKs, illustrating potentially analogous and distinct features.
CRK and the Potential for CaM Activation

The proposed model for CRK activation is based on recent biochemical evidence for CaM activation of some isoforms (42, 100). Although prior studies failed to produce evidence for CRK activation by Ca2+ alone (23, 58), the question of Figure 2 Activation models for a CDPK, CRK, CCaMK, and SnRK3. The structural features of these models are highly speculative. CDPK: Model A is shown as the closest analogy to a CaMK with a exible tether connecting autoinhibitor/junction to the CaM-LD (closed conformation, with empty EF-hands shown as open circles). An alternative model (B) shows the CaM-LD at basal levels of Ca2+ already in a preformed complex with the junction. Activation is proposed to occur when the N-terminal lobe of the CaM-LD binds Ca2+ and relays a conformational change to disengage the autoinhibitor (dotted line). CRK and CCaMKs: The two models are shown to emphasize their potential activation by CaM. The question mark refers to the uncertain role of the tethered C-terminal domain in CRKs (with degenerate EF-hands marked by Xs) and the visinin-like domain in CCaMKs. SnRK3: The autoinhibitor is disengaged by a Ca2+-induced change of CBLs conformation or binding status. The question mark refers to the uncertain relationship of different CBLs with different SnRK3s and the signicance of their role in Ca2+ regulation of kinase activity.
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CaM activation has only been tested recently. An important feature of the CRK model is the presence of a CaM-binding site located between the autoinhibitor and tether, which was experimentally conrmed for OsCBK1 (99) and NtCaMK2 (42). This binding sequence shows reasonable conservation with the analogous CaM-LD-binding site in a CDPK (Figure 3). In CDPKs this sequence facilitates CaM activation, but only when exposed by truncation of the CaM-LD (44). Thus, it seems reasonable that the homologous region of a CRK could function in CaM activation, assuming the binding sequence is exposed and available for interaction. This model raises several questions. For example, what is the function of the CRK C-terminal domain? A function is expected because the C-terminal domain appears well conserved in a comparison of potential rice and Arabidopsis CRK orthologs (typically 80% identity). This is comparable to the conservation between kinase domains and much better conserved than the highly variable N-terminal domains. Thus, the C-terminal end of CRKs may have retained a regulatory function, for example in sensing ions or small molecules other than Ca2+ (new sensor function). Alternatively, this domain may have evolved to provide a nonregulatory function that facilitates the interaction of a CRK with a specic macromolecular complex (targeting function).
CCaMK and Dual Regulation by a Visinin-Like Domain and CaM

The model for CCamK activation is based on studies demonstrating the potential for CaM-stimulated kinase activity. The central feature is that CaM binds to a sequence located between the autoinhibitor and tether (72, 88), analogous to the model for Figure 3 Alignments of regulatory features show differences between CDPKs, CRKs, CCaMKs, and SnRKs. Upper panel: Two regions from the kinase domain show differences of importance to regulation, the activation loop, and an autophosphorylation site in CCaMKs. One representative member of each subfamily from Arabidopsis is presented: CPKs (A to L), CRKs (A to C), PPCKs and PEPRKs (1 and 2), SnRKs1 to 3, along with CCaMKs from rice (Os), lily (Ll), and tobacco (Nt), plus additional sequences for reference. All the sequences are available on the PlantsP website, except for mouse PKA (P05132), Aplysia Twitchin (1KOBA), and rat CaMKI (NP 604463), which were retrieved from Genbank. Bottom Panel: Junction/autoinhibitory domain alignments suggest similarities in the mechanism of autoinhibition and activation in CDPKs, CRKs, and CCamKs. For AtCPK1, the borders are indicated for the Junction and binding domain (iCaM-BD) used for intramolecular interaction with the CaM-LD. Residue F-430 in AtCPK1 functions in autoinhibition (91) and corresponds to a Phe in the autoinhibtor of CaMKI and CaMKII (40). This highly conserved Phe is replaced by a Tyr in the CRKs. The iCaM-BD shows strong conservation in the CRKs and CCaMKs. The autoinhibitory region of Twitchin is shown as an example of variation within the CaMK superfamily. Twitchin is activated by S100 instead of calmodulin. The S100 binding sequence is indicated above. The rat CaMKII Genbank accession number is NP 0037052.
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CRKs. The primary distinction is that CCaMKs have a visinin-like domain that can function as an additional Ca2+-sensor for intramolecular activation (88). This model raises several questions. For example, does the visinin-like domain form a prebound conformation at basal Ca2+ concentrations, as proposed for a CDPK? During CaM activation, does calmodulin displace the visinin-like domain? The junction contains a potential CaM binding sequence that is 17 amino acids longer in CCaMKs than in CRKs and CDPKs (Figure 3), and it may provide an extended target for both intramolecular binding of the visinin-like domain and bi-molecular binding of CaM (or other CaBP).
SnRK3s and the Signicance of Ca2+ Regulation

The model for SnRK3 activation is based on evidence that SnRK3 activity can be stimulated through an interaction with a CBL (2629, 31, 33). All SnRK3s have a NAF/FISL domain that is located immediately C-terminal to the kinase domain and overlaps with a potential autoinhibitory sequence (2, 30). Binding CBLs to the NAF/FISL domain may be Ca2+ dependent (66, 85) or independent (31, 33), depending on the particular kinase or experimental parameters. Activation presumably occurs as a result of the bound CBL disrupting the autoinhibitor. An additional (or alternative?) function for CBL/SnRK3 interactions comes from evidence that AtCBL4 (SOS3) becomes myristoylated and targets SnRK3.11 (SOS2) to a membrane-bound complex (48). This targeting may be subject to Ca2+ regulation, similar to the myristoyl switch features of recoverin (61, 73). Although the model shown here emphasizes the potential role of a CBL in Ca2+ activation, recent studies suggest that other modes of regulation may be more signicant. There are two lines of evidence to support the contention that SnRK3s can be activated by a phosphorylation cascade. First, all SnRK3s have activation loops containing a phosphorylatable Ser/Thr located in the same position shown to regulate the activation of many other kinases (e.g., T-197 for PKA) (1, 65) (Figure 3; Figure 4). Second, activated states were observed in multiple examples of SnRK3 in which the activation loop Ser/Thr was mutated to an Asp to mimic the phosphorylated state (e.g., SnRK3.11, 3.12, 3.13, 3.6 representing SOS2, PKS6, PKS11, and PKS18, respectively) (2628, 31). In SnRK3.11 (SOS2) the activated mutant displayed relatively high constitutive activity that was not further activated by Ca2+ and a CBL. This phospho-mimicked activity was more than tenfold higher than the maximum Ca2+-activated levels of the wild-type SnRK3.11/CBL4 (SOS2/SOS3) complex. Thus, the likelihood of SnRK3 phosphoactivation raises the question of whether the much lower levels of Ca2+-regulated activity are biologically meaningful.
WHY ARE THERE SO MANY DECODERS?

In Arabidopsis there are potentially more than 67 Ca2+-regulated protein kinases. Why so many? Part of that answer includes standard explanations, such as the need to pair different kinase genes with different promoters to facilitate the expression
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of at least one member of a kinase family in many different cell types. The other part is the potential need for specialized biochemical and biological functions, some of which are outlined in the following sections using CDPKs as examples.
Isoform-Specic Ca2+ Activation Thresholds

Multiple isoforms may be needed to decode different Ca2+ signals. Ca2+ signals come in the form of AM [amplitude modulation, e.g., (55)] and FM [frequency modulation, e.g., (3)] signals. Whereas CDPKs are likely involved in decoding FM signals, the mechanism for frequency counting in plants is a matter of speculation. Features of CaMKII in animals provides a useful model for considering mechanisms of frequency decoding (16). For AM signals, different CDPK isoforms can potentially be activated by different levels of Ca2+ (Figure 5). For example, soybean isoforms alpha and gamma display Ca2+ activation thresholds (K-activation) that differ by more than tenfold, with isoform alpha showing a very low threshold around 60 nM Ca2+. Thus, a small-magnitude Ca2+ spike may selectively activate CDPK alpha, and a much higher magnitude spike would activate both alpha and gamma. The structural explanation for how different CDPKs sense different Ca2+ thresholds is not clear. The simplest explanation is that different CaM-LDs evolved with divergent EF-hands with different Ca2+-binding afnities. However, this explanation has not been tested and may not be the sole component of Ca2+ threshold variations. Threshold variation may also be related to the interesting observation that different peptide substrates can alter the Ca2+ activation thresholds by more than tenfold. We refer to this as substrate modulation (Figure 5). Substrate modulation
Figure 5 Ca2+ activation thresholds can vary depending on CDPK isoform and substrate (adapted from Lee et al., 56). In a comparison between the soybean alpha and gamma isoforms, calcium activation thresholds show a difference of more than tenfold, using the same syntide-2 as a substrate. However, these thresholds can vary depending on substrate. In the graph to the right, the soybean isoform alpha displays a more than tenfold-weaker Ca2+ sensitivity when the substrate changed from syntide to histone.
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likely results from the interconnected relationship of different domains. An example of this interconnectedness is shown by a dramatic 100-fold increase in Ca2+ afnity when a CaM-LD interacts with the junction domain (J. Christodoulou, J. Harper & W. Chazin, unpublished data), analogous to the changes in Ca2+ afnity observed when CaM interacts with a binding sequence (8, 64). Thus, changes in other features of the kinase, besides the EF-hands, may also contribute to isoformspecic activation thresholds. Regardless of the mechanism, an important message is that every CDPK-substrate complex has the potential to respond to different AM Ca2+ signals.
Phospho-Regulation
Multiple isoforms may be needed to provide nodes of cross-talk with other phosphosignaling pathways. Many kinases are activated through autophosphorylation or a kinase cascade. For example, PKA (protein kinase A) is activated by phosphorylation at T-197 (Figures 3, 4) (4, 65). The equivalent site in a typical CDPK and CRK is an Asp or Glu (D-310 in AtCPK1) that has been proposed to mimic a phosphoactivated state. In contrast, this site in SnRKs1, 2, or 3 is a Ser or Thr (Figure 3), consistent with the hypothesis that SnRKs are potentially phospho-regulated. This suggests that unlike SnRKs, most CDPK activity occurs independently of an activation loop phosphorylation. However, there is evidence for phospho-regulation of at least one CDPK (NtCDPK2) (76, 77). A trans-phosphorylation event (i.e., not autophosphorylation) results in a 10- to 250-fold-increase in maximum Ca2+-stimulated activity, as assayed in immuno-precipitates of a transiently expressed epitope-tagged kinase. This phospho-activation occurs in response to certain pathogens, possibly through MAPK (mitogen-activated protein kinase) cascades. However, the upstream kinase and site of phospho-regulation have not been determined. In addition, a rice membraneassociated CDPK is activated post-translationally in response to cold stress (63). Autophosphorylation of some CDPKs occurs by an intramolecular mechanism at submicromolar concentrations of Ca2+ (12, 36, 56, 79). For AtCPK1, autophosphorylation is likely required for 143-3 binding and stimulation (11). Autophosphorylation was observed as a prerequisite for substrate phosphorylation by a CDPK from groundnut (12), but inhibitory for CDPK isolated from winged bean (79). Autophosphorylation did not render soybean and groundnut CDPKs insensitive to Ca2+ (12, 36), as has been observed for animal CaMKII (16). However, autophosphorylation of a CRK (NtCaMKI), which requires Ca2+/CaM, leads to an active enzyme with activity independent of Ca2+/CaM (100). In CCaMKs, autophosphorylation results in an increased afnity for CaM and is necessary for maximal CaM activation (82). The autophosphorylation site in lily CCaMK was determined to be T267 by mass spectrometry, and is near subdomain X (83) (see Figure 3). A T267A mutant did not autophosphorylate and it was no longer stimulated by Ca2+/CaM. This CCaMK autophosphorylation site is not conserved in CDPKs.
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Thus, the potentially diverse consequences of autophosphorylation clearly need to be examined for all Ca2+-regulated kinases.
Lipid Regulation
Multiple isoforms may be needed to provide nodes of cross-talk with lipid-signaling pathways. Although evidence for phospho-lipid-stimulated kinase activity has been reported from plants, it is not clear if there is a true homologue of an animal PKC (protein kinase C), or if other kinases, including CDPKs, provide functionally analogous activities. A distinguishing feature of PKC is the presence of a C2domain that promotes binding of the kinase to membranes in the presence of Ca2+. Although no Arabidopsis kinases contain C2 domains (PlantsP domain search), a CDPK from the green alga Dunaliella, DtCPK1, does have an N-terminal C2domain (71). And although the closest relatives of PKC in Arabidopsis are likely encoded by At3g08720 and At3g08730 (92), their biochemical activities have not been reported. AtCPK1 is stimulated by lysophosphatidylcholine and several phosphatidylinositols (PI, PIP, PIP2), but not inositol, inositol triphosphate, phosphatidylcholine, or phosphotidylserine. In contrast to AtCPK1, a carrot CDPK is activated by phosphotidylserine (20), as is a putative CDPK from maize ZmCPK11 (J. Szczegielniak & G. Muszynska, personal communication; 87). ZmCPK11 is also slightly stimulated (1.5-fold) by diacylglycerol. Regardless of the degree of overlap between CDPK and classic PKC activities, the important message is that lipid-signaling pathways need to be considered as potential regulators of CDPKs.
Subcellular Targeting and Isoform-Specic Complexes

Multiple isoforms may be needed to provide Ca2+-regulated activity at specic subcellular locations, or within specic protein complexes. CDPKs are targeted to multiple locations, including the cytosol, nucleus, plasma membrane, endoplasmic reticulum, peroxisomes, mitochondrial outer membrane, oil bodies, and through plasmodesmata into sieve tubes (5, 14, 60, 67, 70, 98). At present, the 26S proteasome (57) represents the rst example of a CDPK in a specic protein-protein complex. However, we expect that the variable N-terminal domains of CDPKs will provide a diverse set of docking sites to facilitate many different isoform-specic protein complexes.
Substrate Specicity
Multiple isoforms may be needed to provide isoform-specic phosphorylation specicities, much like different families of kinases show clear substrate specicities. For example, CDPKs do not appear to recognize the same substrate sites as SnRK3, which prefers peptide substrates of SnF1 kinase (2630, 85); casein kinase, which prefers acidic residue at P + 4 (69); and MAPK, which prefers a proline at P + 1 (15).
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However, within the CDPK family, there is no evidence yet for any dramatic differences in isoform-specic substrate specicity. Consistent with this observation, an in silico survey of the Arabidopsis and rice CDPKs suggests a strong conservation of 13 residues predicted to contribute to substrate specicity, as indicated by crystal structures of related kinase (Figure 4) that show the potential of specic residues to interact with the substrate from the site of phosphorylation (P) upstream to P-5 (G. Breton, A. Harmon & J. Harper, unpublished data). Nevertheless, very subtle changes in structure and alternative substrate interaction sites may provide a yet undetected diversity of substrate specicities. A global analysis of potential substrates is needed.
COMPARATIVE KIN-OMICS
The availability of genome sequences for both rice and Arabidopsis provides the rst opportunity to make a comprehensive comparison of gene families between a monocot and eudicot. This has begun for the CDPKs from Arabidopsis (34 isoforms) and rice (27 isoforms) (G. Breton & J. Harper, unpublished data).
Twelve Subfamilies of CDPKs in Flowering Plants

A phylogenic comparison of kinase domains and regulatory domains provides the basis for delineating at least 12 subfamilies of CDPKs, 3 subfamilies of CRKs, 1 family of PPCKs, 2 subfamilies of PEPRKs, and 1 family of CCaMKs (Figure 6). The Arabidopsis isoforms that belong to these subfamilies can be viewed in Figure 7. These 19 subfamilies are present in the ancestor of monocots and eudicots. However, the CCaMKs have not been found in Arabidopsis. This loss appears to be an evolutionarily recent event, as CCaMKs have been found in another eudicot (e.g., tobacco). Nevertheless, the apparent conservation of 18 subfamilies during the evolution of monocots and eudicots supports a hypothesis that each subfamily provides a necessary biological function for owering plants.
Regulatory Features
From the alignment of kinase domains, the phospho-regulatory site of PKA (T197) aligns with a potential phospho-mimic residue in CDPKs and CRKs (D-310 in AtCPK1) (Figure 3). This phospho-mimic is proposed to make CDPK activity independent of autophosphorylation or a phospho-activating kinase cascade. However, there are three CDPK isoforms (AtCPK 22 and 31 and OsCPK-J2) that provide an exception to the rule, harboring an Arg, Lys, or Gln at this position, respectively. Similarly, CCaMKs have an Ala or Gly at this position, which further supports their having a distinct set of regulatory features.
The Importance of Species-Specic Differences

Is the species-specic variation among the plant kinases and their substrates important, or does it represent evolutionary noise? One hypothesis is that changes
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Figure 6 Nineteen subfamilies are delineated by sequence homology-based clustering of the Arabidopsis and rice CDPKs, CRKs, and CCaMKs. Figure 7 shows the Arabidopsis genes corresponding to each subfamily. The sequence corresponding to the catalytic (Kinase Domain tree) and CaM-like domain (CaM-LD tree) were aligned separately using ClustalW (G. Breton & J. Harper, unpublished data) from sequences obtained from PlantsP. The neighbor joining trees shown were constructed using MEGA2 (54). The bootstrap values are shown at the node of each branch. The two trees agree and provide independent evidence for the subfamily designations shown to the left (e.g., CPK subfamilies A to L). The number of isoforms in each subfamily for rice (Os) and Arabidopsis (AT) are listed next to the subfamily designation. As a reference, one member of each subfamily is listed on top of the corresponding kinase domain model. This tree was constructed using the following outliers (not shown): rat CaMKI (NP 604463), Drosophila CaM (NP 725120), and rat Hippocalcin (visinin-like) (NP 058818). Triangles represent intron position and X represents the absence of an intron.
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in signal transduction pathways provide a signicant source of variation needed to evolve plants with unique morphologies and ecological adaptations. Thus, comparative kin-omics promises to help identify features of kinases and substrates that may help explain the tremendous diversity of form and function within the plant kingdom. From this perspective, four examples illustrate interesting features of the CDPK/ SnRK superfamily that stand out: (a) the apparent absence of a CCaMK in Arabidopsis (Figure 6); (b) the relative expansion of the CDPK-G subfamily in Arabidopsis with nine representatives compared to one in rice (Figure 6); (c) in the Arabidopsis CDPK subfamily G, isoforms AtCPK22 and 31 currently provide the only examples of CDPKs with a basic residue instead of an acidic residue in the equivalent position to the phospho-regulatory site within the activation loop; and (d) the phosphorylation sites identied in certain substrates are not necessarily conserved in corresponding homologs from other species, such as IKLDKMPS378FTMD in the serine acetyl transferase from soybean GmSAT1 (F. Liu, B.-C. Yoo & A.C. Harmon, unpublished data) and KRRFRFTANLS45KR in the Ca2+ pump ACA2 from Arabidopsis (44, 47). Although the absence of a CCaMK in Arabidopsis indicates that this gene is not essential for all plants, it is equally likely that such kinases still provide unique regulatory features for the species that harbor them. Similarly, whereas the expansion of one gene family may reect an accident of redundant gene duplication, duplications may provide templates for the evolution of new kinase features with species-specic importance. For example, kinases such as CDPK22 and 31 with altered activation loops may provide new regulatory features of importance to an Arabidopsis-specic signaling pathway. Finally, the divergence of phosphorylation sites in substrates is clearly a powerful evolutionary mechanism to alter the kinetics of physiological important pathways, and thus alter the form and function of plant species or ecotypes. Ultimately, we need to understand both the conserved features and species-specic variations in phospho-signaling networks.
Expression Proling
A detailed picture of every genes expression pattern is expected to emerge over the next few years. Figure 7 provides the relative expression levels for the Arabidopsis CDPKs in roots, shoots, and pollen. At least one representative of each subfamily is expressed in either roots, shoots, or pollen. The root-shoot expression proles show detectable expression for representatives of 10 of the 12 subfamilies. In most cases the root expression levels were similar or higher than the shoot expression levels. Only 6 of the CDPKs appeared undetectable in this survey. It is not clear if the low-level expression of these genes is a consequence of restricted expression in rare cell types or specic times in development, or if it represents a subset of weakly expressed isoforms. An important observation is that pollen show detectable expression for at least one representative CDPK in 8 of the 12 subfamilies. The pollen expression levels
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appear very high for 5 CDPKs (CPK14, 16, 17, 24, and 34). Although this pollen prole currently provides the only dataset on an isolated cell type from plants, it supports a working hypothesis that a typical plant cell expresses CDPKs from 8 or more different subfamilies.
Biological Functions
A growing list of observations supports the hypothesis that different CDPKs have different functions, including the following. The rst evidence of an essential role for CDPKs was obtained through oligo-based silencing of CDPKs in maize pollen (18). In maize leaf protoplasts, transient overexpression of constitutively active AtCPK10 or AtCPK30 caused the activation of the stress-inducible HVA1 promoter (84). In rice, overexpression of OsCPK7 made the transgenic lines more tolerant to cold, drought, and salt-stress (80), and antisense expression of a CDPK called SPK resulted in defective accumulation of starch (6). In tomato protoplasts, overexpression of AtCPK1 enhanced activity of NADPH oxidase and oxidative burst (96). In tobacco, a virus-induced gene silencing method (VIGS) used to silence CDPKs reduced and delayed the hypersensitive response to fungal Avr9 elicitor (76). In newly emerged leaves of tobacco, VIGS silencing of NtCDPK1 resulted in abnormal cell morphology and cell death (57). In Arabidopsis, an embryo lethal phenotype was reported for two independent KO alleles of CPK28 (C. Chan & M. Sussman, personal communication). An equally diverse list of functions is expected for the CRKs and SnRKs. The emerging use of knockouts to study the different Arabidopsis kinases offers a powerful approach to elucidating the essential and subtle features of each subfamily.
A REASONABLE WAY TO THINK ABOUT Ca2+-REGULATED KINASES

Although it is reasonable to emphasize the exciting aspects of Ca2+ signals in phospho-regulation, the diagram of CDPK signaling in Figure 8 provides a reminder of additional regulatory features to consider, and the potential of these features to feed back on each other. As Figure 5 illustrates, even the interaction of a kinase with its substrate can change the enzymes Ca2+ activation threshold.
Does Reasonable Evidence for Ca2+ Regulation Exist?

Presently, the best evidence that CDPKs function in Ca2+ signaling is based on their potential to be activated by Ca2+, as shown through in vitro biochemistry. A typical CDPK is stimulated 10- to 100-fold by increasing Ca2+ from nM to low M concentrations. This range of Ca2+ is consistent with expected changes during in vivo Ca2+ signaling. At maximal activation, CDPKs have specic activities in the range of 1 to 4 M/min/mg (37), consistent with the Vmax of other typical kinases such as CaMKII, PKA, and PKC (17).
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Figure 8 The CDPK signal transduction pathway shows the many points at which the output can be modied.
Although careful in vitro biochemistry provides a foundation for predicting in vivo activity, it is possible to obtain misleading results when enzymes are assayed under nonphysiological conditions. A potential pitfall in the assay of Ca2+regulated enzymes is to only conduct assays under the extremes of plus and minus Ca2+ (e.g., in 1 mM Ca2+ versus 10 mM EGTA), which changes Ca2+ concentrations from levels far below to far above normal physiological levels, and may cause signicant changes in magnesium concentrations as well. It is possible that these extremes will yield activity differences that would never occur in vivo and are not related to Ca2+ signaling. Nevertheless, we still have a poor understanding of in vivo conditions, and extremes may be the norm for certain microenvironments. Thus, although it is reasonable to consider any Ca2+-regulated change as potentially important, it is prudent to be cautious when speculating on potential signaling functions.
For Every Ca2+ Signal, CDPKs are Expected to Phosphorylate Many Targets
The rationale for this speculation is the following: (a) A typical CDPK is activated 10- to 100-fold by Ca2+ in the low M range (74). (b) CDPKs are found throughout the cytoplasm, in the nucleus, and bound to multiple membrane systems (14). (c) CDPKs can phosphorylate many sequences with Kms in the low M range, including those containing the general consensus of Basic-X-X-S/T or [Basic-Basic-X-Basic]-Hydrophobic-X(4)-S/T-X-Basic (45, 46). (d) No dramatic substrate specicity differences have yet been found for CDPK isoforms. Therefore, it is reasonable to hypothesize that a substrate will be phosphorylated in response to a Ca2+ signal in planta if the in vitro biochemistry has established a low M Km for its phosphorylation by a generic CDPK. There is a growing list of potential substrates that was recently reviewed (13), some of which have phospho-dependent activity changes. The working model for CDPKs is that some isoforms are multifunctional (with many potential substrates,
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similar to CaMKII in animals) and others may be dedicated as part of a complex, with one or few substrate targets (similar to MLCK, myosin light chain kinase, in animals).
CONFIRMING REGULATORY NETWORKS IN PLANTA

A long-term goal is to understand the in vivo function of every kinase and phosphatase in a model plant. This includes (a) identifying which phosphorylation sites have regulatory importance, and (b) quantitating changes in every phosphorylation site in response to developmental or environmental stimuli. An immediate challenge is to adapt current technologies for application to the entire proteome. Within this challenge, we offer a perspective on two potential cornerstone technologies.
Phospho-Mimic Mutations
We expect an explosion in the number of experimentally veried phosphorylation sites in plant proteins as a result of rapid advances in mass spectrometry (22, 24, 62). However, it is likely that a relatively small fraction of these phosphorylation sites will have a signicant functional importance. A key question is how to identify the most important phospho-regulatory sites. In some cases it will be possible to obtain functional inference through a genetic approach, for example by mutating phosphorylation sites to an Ala (which provides a nonphosphorylatable control) or an Asp or Glu. The Asp or Glu provides a negative charge that can sometimes mimic a phosphorylated site. The mutant proteins can then be tested both in vitro and in vivo for functional changes. Using Arabidopsis, it is reasonable to suggest testing the function of each target protein as a nonphosphorylatable or phospho-mimic mutant. This could be done in a plant-host background in which the endogenous target gene was knocked out. This would allow the mutant protein to be tested in the absence of wildtype protein. However, given the potential that most proteins will have multiple phosphorylation sites, there is a need to invest in developing a bioinformatics approach to help prioritize experimental efforts.
Phospho-Site-Specic Antibodies
Understanding signal transduction will also require a quantitative understanding of phosphorylation changes. The phosphorylation status of a given target results from the balance between opposing kinase and phosphatase activities. High-throughput methods will be needed to monitor the dynamics of every phospho-regulatory site. There is currently an underutilized potential to generate antibodies that specifically recognize a phosphorylated sequence. Such antibodies can be used to distinguish between phosphorylated and unphosphorylated states of specic proteins (9). A standard approach to generating such antibodies is to synthesize a peptide
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containing the phospho-site of interest and couple it to keyhole limpet hemocyanin for immunization of rabbits. Phospo-site-specic antibodies are puried by sequential chromatography on resins of immobilized dephosphopeptide (to remove antibodies that recognize the peptide, but not the phosphopeptide) and phosphopeptide (to isolate antibodies that bind only the phospho-peptide). It is important to also have antibodies that recognize total protein (unphosphorylated plus phosphorylated) so that effects on protein abundance will not be mistaken for changes in phosphorylation state. Although there are many commercially available target-site-specic antibodies, none are available for plant-specic proteins. For research on the plant Ca2+ signaling networks, there are three examples of the phospho-site-specic antibody approach. Komina et al. (52) developed antibodies directed to the site in sucrose synthase from soybean root nodules that is phosphorylated by nodule CDPK (101, 102). Using the phospho-specic antibodies and antibodies that recognize total sucrose synthase, there was a decrease in the abundance and phosphorylation state of the enzyme in stress conditions. From their results they speculated that phospho-sucrose synthase may be protected from degradation. In the second example, Tang et al. (89) used antibodies directed against two sites in pyruvate kinase from soybean to show that both sites are phosphorylated in vivo. One site is proposed to be phosphorylated by CDPK and the other by a SnRK1. The data suggest that phosphorylation leads to an increase in proteolytic degradation of the pyruvate kinase, in contrast to a protective role of phosphorylation on nodule sucrose synthase. In the third example an antibody was raised against a CDPK phosphorylation site located in a soybean serine acetyltransferase SAT1 (F. Liu & A. Harmon, unpublished data). Phosphorylation of this site blocks feedback inhibition of the enzyme by cysteine and thus stimulates the cysteine production from serine. An increase in cysteine level promotes the synthesis of homoglutathione, which is important for some biotic and abiotic stress response pathways. By using this site-specic antibody, it was possible to demonstrate an increase in phosphorylation of SAT1 in response to a stimulus that produces a cytosolic Ca2+ signal. It is reasonable to propose an increased effort to develop phospho-site-specic antibodies to help quantitate the dynamics of the plant phosphorylome. A large antibody collection could be used in chip-array format to simultaneously detect the phosphorylation levels at thousands of specic sites. However, innovation is needed to make the production of large numbers of state-specic antibodies economically feasible for the plant community. Thus, it is encouraging that progress is being made to adapt mass spectrometry to quantitate phosphorylome dynamics (24). Nevertheless, the efcacy of mass spectrometry has not yet been proven as a high-throughput strategy for averageand low-abundance proteins. In addition, an important limitation is the difculty in routinely obtaining 100% sequence coverage in the analysis of most proteins, especially those with low abundance. Thus, it is likely that phospho-site-specic antibodies will provide a powerful complementary approach for the foreseeable future.
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A FUTURE PERSPECTIVE
A roadmap to the plant phosphorylome is beginning to emerge. At the center is the goal of a computer model that integrates the complex interactions between more than 1000 kinases, 100-plus phosphatases, and thousands of substrates. This is a daunting task, even for a smaller subset of 67 potential Ca2+-regulated kinases in Arabidopsis. In the near future, there is a realistic expectation that Arabidopsis can provide a useful paradigm for the CDPK-SnRK superfamily. This will include genetic evidence for biological function through the analysis of null and dominant mutations. However, Arabidopsis is only a starting point. It is likely that species-specic variations in signal transduction, such as Ca2+-mediated phosphoregulation, result in fundamental differences that make one plant species different from another. The emerging eld of comparative kin-omics has already revealed interesting species-specic differences in CDPKs and their substrates. Understanding the role of this variation may provide the basis for engineering crop plants with modied signaling pathways of importance to commercially important traits. ACKNOWLEDGMENTS We thank the NSF for support to J.H. and A.H. (DBI-99,75808), the Fonds de Recherche sur la Nature et les Technologies for a postdoctoral fellowship to G.B., and the NASC website for making transcriptome results publicly available.
The Annual Review of Plant Biology is online at http://plant.annualreviews.org
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C-1
Figure 4 Kinase structures showing different modes of interaction with pseudosubstrate inhibitory sequences. The inhibitory peptide of protein kinase A (A) and autoinhibitory segments of CaMKI (B) and Twitchin (C) are shown as red worms. Each inhibitor contains a motif that is similar to the preferred phosphorylation sites in substrates, but that does not contain a phosphorylatable amino acid. The inhibitor peptides and their pseudosubstrate sequences are thought to interact with the kinases in a manner similar to substrates (25, 50, 51). Residues of the pseudosubstrate sequence are shown as balls and sticks and are labeled. P is the residue at the position equivalent to the phosphorylatable amino acid. Residues C-terminal and N-terminal to P are numbered with negative and positive numbers, respectively, in order of proximity from P. P-loops (residues in subdomain I that interact with ATP) and activation loops (sequence between subdomain VII and VIII) are shown as cyan and yellow worms, respectively. The activation loop of CaMKI is disordered and is shown as a yellow dotted line, and a second disordered region is shown as a white dotted line. Phosphothreonine-197 in the activation loop of PKA, which is essential for the activation, is shown by a gray ball and stick that interrupts the yellow activation loop. The catalytic Asp residue of the DL/IKPEN sequence in subdomain VIb is shown as blue balls and sticks. The amino terminal end of each (auto)inhibitor interacts with the large lobe of the kinase, but interaction of the C-terminal ends differ dramatically. In PKA and Twitchin the pseuodosubstrate sequence threads through the active sites, whereas for CaMKI it interacts mostly with the P-loop. The structures were drawn in Protein Explorer (http://molvis. sdsc.edu/protexpl/frntdoor.htm) using coordinates deposited in the Brookhaven Protein Database for protein kinase A (1ATP), CaMKI (1A06), and Twitchin (1KOB).
C-2 HARPER
s
BRETON
s
HARMON
See legend on next page
C-3
Figure 7 Relative expression levels of the Arabidopsis CDPK family in root, shoot, and pollen suggest that members of 14 out of 18 subfamilies are expressed in pollen, and 16 out of 18 families are detectable in either vegetative root or shoot tissues. To provide a comparison of data from different oligo arrays done in different labs, the data were normalized to the total expression levels in a particular tissue for members of the entire CDPK-SnRK superfamily, and reported here on a scale of parts per 10,000. Root (white bars) and shoot (green bars) data are from I. Baxter & J. Harper (unpublished data). They represent the average of 12 experiments under different growth conditions (3 h, 27 h, and 75 h with fresh media, or salt (100 mM), or cold (4C), or mannitol (200 mM)). Error bars represent the standard deviation. Pollen data from Honys & Twell were obtained from the NASC website [(39) and http://ssbdjc2.nottingham.ac.uk/narrays/experimentbrowse.pl]. Tricellular pollen (blue bars) and mature pollen (yellow bars) are the average of duplicated experiments. Error bars represent the range. A comparison of tricellular and mature pollen provides independent support for the relatively high expression levels for more than five CDPKs. The white and black boxes below the x-axis indicate undetectable or detectable levels of expression, respectively. The absence of a box indicates that no oligo set was available to detect this gene.
Annual Review of Plant Biology Volume 55, 2004
CONTENTS
AN UNFORESEEN VOYAGE TO THE WORLD OF PHYTOCHROMES,
Masaki Furuya
ALTERNATIVE NAD(P)H DEHYDROGENASES OF PLANT MITOCHONDRIA, Allan G. Rasmusson, Kathleen L. Soole,

and Thomas E. Elthon 23 41
DNA METHYLATION AND EPIGENETICS, Judith Bender PHOSPHOENOLPYRUVATE CARBOXYLASE: A NEW ERA OF STRUCTURAL BIOLOGY, Katsura Izui, Hiroyoshi Matsumura,
Tsuyoshi Furumoto, and Yasushi Kai
69 85
METABOLIC CHANNELING IN PLANTS, Brenda S.J. Winkel RHAMNOGALACTURONAN II: STRUCTURE AND FUNCTION OF A BORATE CROSS-LINKED CELL WALL PECTIC POLYSACCHARIDE,
Malcolm A. ONeill, Tadashi Ishii, Peter Albersheim, and Alan G. Darvill
109
NATURALLY OCCURRING GENETIC VARIATION IN ARABIDOPSIS THALIANA, Maarten Koornneef, Carlos Alonso-Blanco, and
Dick Vreugdenhil 141
SINGLE-CELL C4 PHOTOSYNTHESIS VERSUS THE DUAL-CELL (KRANZ) PARADIGM, Gerald E. Edwards, Vincent R. Franceschi,
and Elena V. Voznesenskaya 173 197 225 263 289 315
MOLECULAR MECHANISM OF GIBBERELLIN SIGNALING IN PLANTS,

Tai-ping Sun and Frank Gubler
PHYTOESTROGENS, Richard A. Dixon DECODING Ca2+ SIGNALS THROUGH PLANT PROTEIN KINASES,
Jeffrey F. Harper, Ghislain Breton, and Alice Harmon
PLASTID TRANSFORMATION IN HIGHER PLANTS, Pal Maliga SYMBIOSES OF GRASSES WITH SEEDBORNE FUNGAL ENDOPHYTES,
Christopher L. Schardl, Adrian Leuchtmann, Martin J. Spiering
TRANSPORT MECHANISMS FOR ORGANIC FORMS OF CARBON AND NITROGEN BETWEEN SOURCE AND SINK, Sylvie Lalonde,
Daniel Wipf, and Wolf B. Frommer 341
vii
viii
CONTENTS
REACTIVE OXYGEN SPECIES: METABOLISM, OXIDATIVE STRESS, AND SIGNAL TRANSDUCTION, Klaus Apel and Heribert Hirt THE GENERATION OF Ca2+ SIGNALS IN PLANTS,
Alistair M. Hetherington and Colin Brownlee
373 401 429
BIOSYNTHESIS AND ACCUMULATION OF STEROLS, Pierre Benveniste HOW DO CROP PLANTS TOLERATE ACID SOILS? MECHANISMS OF ALUMINUM TOLERANCE AND PHOSPHOROUS EFFICIENCY,
eros Leon V. Kochian, Owen A. Hoekenga, and Miguel A. Pin
459 495 521 537 555
VIGS VECTORS FOR GENE SLIENCING: MANY TARGETS, MANY TOOLS, Dominique Robertson GENETIC REGULATION OF TIME TO FLOWER IN ARABIDOPSIS THALIANA,
Yoshibumi Komeda
VISUALIZING CHROMOSOME STRUCTURE/ORGANIZATION,

Eric Lam, Naohiro Kato, and Koichi Watanabe
THE UBIQUITIN 26S PROTEASOME PROTEOLYTIC PATHWAY,

Jan Smalle and Richard D. Vierstra
RISING ATMOSPHERIC CARBON DIOXIDE: PLANTS FACE THE FUTURE,

Stephen P. Long, Elizabeth A. Ainsworth, Alistair Rogers, and Donald R. Ort 591
INDEXES
Subject Index Cumulative Index of Contributing Authors, Volumes 4555 Cumulative Index of Chapter Titles, Volumes 4555 629 661 666
ERRATA
An online log of corrections to Annual Review of Plant Biology chapters may be found at http://plant.annualreviews.org/

D Ca S T P P K: Ecoding 2+ Ignals Hrough Lant Rotein Inases

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Annu. Rev. Plant Biol. 2004. 55:26388 doi: 10.1146/annurev.arplant.55.031903.141627 Copyright c 2004 by Annual Reviews.

DECODING Ca2+ SIGNALS THROUGH PLANT PROTEIN KINASES

Nomenclature: A Very Good Place to Start

CCaMK, Ca2+ AND CALMODULIN-ACTIVATED KINASES

CBL, CALCINEURIN B-LIKE Ca2+-BINDING PROTEIN

SENSOR RESPONDER VERSUS SENSOR RELAY

MODELS FOR Ca2+ REGULATION The CDPK Paradigm

CRK and the Potential for CaM Activation

CCaMK and Dual Regulation by a Visinin-Like Domain and CaM

SnRK3s and the Signicance of Ca2+ Regulation

WHY ARE THERE SO MANY DECODERS?

Isoform-Specic Ca2+ Activation Thresholds

Subcellular Targeting and Isoform-Specic Complexes

Twelve Subfamilies of CDPKs in Flowering Plants

The Importance of Species-Specic Differences

A REASONABLE WAY TO THINK ABOUT Ca2+-REGULATED KINASES

Does Reasonable Evidence for Ca2+ Regulation Exist?

CONFIRMING REGULATORY NETWORKS IN PLANTA

See legend on next page

Annual Review of Plant Biology Volume 55, 2004

ALTERNATIVE NAD(P)H DEHYDROGENASES OF PLANT MITOCHONDRIA, Allan G. Rasmusson, Kathleen L. Soole,

MOLECULAR MECHANISM OF GIBBERELLIN SIGNALING IN PLANTS,

373 401 429

459 495 521 537 555

VISUALIZING CHROMOSOME STRUCTURE/ORGANIZATION,

THE UBIQUITIN 26S PROTEASOME PROTEOLYTIC PATHWAY,

RISING ATMOSPHERIC CARBON DIOXIDE: PLANTS FACE THE FUTURE,

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