BACKGROUND
one of the significant factors determining successful plant growth and reproduction is an efficient defense against insect attacks.
after herbivore feeding there is a dramatic ca2+ influx limited to a few cell layers lining the damage zone  <cit> .
signals induced rapidly by herbivore attack have been found to spread over the leaf, leading to a strong ca2+-dependent transmembrane potential  depolarization in the damage zone followed by a transient vm hyperpolarization in the close vicinity and a constant depolarization at distances greater than 6- <dig> mm  <cit> .
these initial cues are transmitted within the plant by signal transduction pathways that include phosphorylation cascades, such as mitogen-activated protein  kinases, and the jasmonic acid  pathway, which play a central and conserved role in promoting resistance to a broad spectrum of insects  <cit> .
however, there is a missing link to downstream signaling and gene regulation for defense responses.
in this network, ca2+-binding sensory proteins are of particular interest, since following ca2+ influx the sensory proteins may secondarily decode information contained in the temporal and spatial patterns of the signal trafficking to control metabolism and gene expression  <cit> .
plants possess several classes of ca2+-binding sensory proteins, including calmodulins, calmodulin-like proteins, calcineurin b-like proteins, and ca2+-dependent protein kinases   <cit> .
the cpks are of special interest, since they represent a novel class of ca2+ sensors, having both a protein kinase domain and a calmodulin-like domain  in a single polypeptide  <cit> .
cpks constitute a large family of serine/threonine protein kinases that are broadly distributed in the plant kingdom.
for example, the arabidopsis genome is predicted to have  <dig> different cpks  <cit> .
arabidopsis cpk <dig> was the first cpk to be characterized, and is known to be activated by phospholipids and 14-3- <dig> proteins, which are small, highly conserved eukaryotic proteins that regulate multiple cellular enzymes, including protein kinases  <cit> .
atcpks  <dig>   <dig>   <dig>   <dig> and  <dig> act as abscisic acid  signaling components, and are involved in aba-responsive gene expression, seed germination, seedling growth, and stomatal movement  <cit> .
especially, atcpks  <dig>   <dig> and  <dig> are likely to interact with aba-related leucine zipper class transcription factors  <cit> , indicating the proximate involvement of cpks in transcriptional regulation.
curiously, little attention has been given to the role of cpks in defense responses.
only one case has been reported: in tobacco, ntcdpk <dig> modulates the activation of stress-induced map kinases, and this interaction requires the synthesis and perception of wound hormones  <cit> .
the role of cpks involved in the defense response against insect herbivory has never been reported.
in this report, we show the involvement of two arabidopsis cpks  in the herbivory-induced signaling network via post-translational regulation of the defense-related transcriptional machinery.
implications for possible signal trafficking via cpks are discussed.
RESULTS
cpk <dig> and cpk <dig> mutants showed decreased transcript levels of defense genes in s. littoralis-damaged leaves
we obtained  <dig> t-dna insertion mutant lines that were putative atcpk-deficient mutants from the european arabidopsis stock centre.
homozygous t-dna insertion lines corresponding to each cpk gene were confirmed using the pcr method  and challenged with herbivore damage.
transcript levels of plant defensin gene pdf <dig> , which is induced in wild-type  leaves exposed to spodoptera littoralis larvae for  <dig> h, were investigated in the mutant leaves .
compared to s. littoralis-damaged wt leaves, t-dna insertion line cpk <dig>  and two different cpk <dig> lines  and cpk13- <dig> ) showed significantly lower transcript levels in s. littoralis-damaged leaves.
the molecular analysis of cpk t-dna insertion mutants and gene expression profiling of the mutants are shown in additional file  <dig>  in addition to our study of pdf <dig> , the time-courses of the transcript levels of two other defense-related genes  after herbivore attack were examined in cpk <dig>  cpk13- <dig>  and cpk13- <dig> leaves and compared to those in wt leaves .
the transcript level of thi <dig>  in wt leaves was increased about  <dig> fold after  <dig> h but declined after  <dig> h. the cpk <dig> mutant showed a comparable level of the thi <dig>  expression during the time course, whereas the transcript levels in cpk13- <dig> and cpk13- <dig> leaves remained unchanged at  <dig> h, but increased after  <dig> h. thus, wt and cpk <dig> probably have different temporal regulation.
throughout the time-course, the induced transcript levels of vsp <dig> were comparable between the wt and all the mutants .
possible involvement of phytohormone signaling in the herbivory-related cpk cascades
to assess whether loss of cpk function affects the signal transduction involved in the defense response, we explored the biosynthesis of ja, jasmonyl-l-isoleucine , ethylene, and abscisic acid .
as shown in figure  <dig>  all the phytohormones examined in this study were formed and accumulated at a similar rate in the infested wt, compared to the cpk mutant leaves , indicating that cpk <dig> and cpk <dig> are not upstream signal kinases for the biosynthesis of ja, aba and ethylene.
we also investigated the pdf <dig>  expression levels in wt and in cpk <dig> and cpk <dig> mutants by applying an exogenous solution of either ja, ethephon , or aba .
compared to the control, after  <dig> h the transcript levels of pdf <dig>  were very slightly induced by ja or aba to levels comparable to those in wt and mutant leaves.
ethephon treatment resulted in drastic but comparable increases of the transcript in both wt and mutant leaves.
we therefore conclude that the cpk <dig> and cpk <dig> cascades are not closely linked to the above phytohormone signaling and biosynthesis cascades.
increased intracellular ca2+ levels in cpk <dig> leaf cells after herbivore and mechanical damage
it is interesting to note that the cpk <dig> mutant showed abnormal changes of the cytosolic ca2+ level after insect damage.
as shown in figure  <dig>  when the membrane-permeable fluo- <dig> am  was applied to wt leaf tissues, it showed a cytoplasmic subcellular localization at sites damaged by s. littoralis  or after mechanical wounding .
cpk <dig> but not cpk13- <dig> plants showed a more marked increase of the cytosolic ca2+ level after the damage, compared to wt.
substrate targeting of cpk <dig> and cpk13
in previous studies, cpk <dig> was found to be localized in the nucleus and the cytosol  <cit> .
in this study, we additionally tested the subcellular localization of cpk13-gfp fusion proteins in transiently expressing onion peels, which revealed nuclear, cytosolic and plasma membrane localizations of the fusion proteins .
accordingly, in order to screen the protein target of cpk <dig> and cpk <dig>  protein kinase assays with  <dig> nuclear and cytosolic protein substrates synthesized using the wheat germ cell-free system  were carried out.
cpk <dig> or cpk <dig> proteins purified from a cell-free extract were incubated with radiolabeled atp, cacl <dig> and the  <dig> distinct substrates.
both cpk <dig> and cpk <dig> showed auto-phosphorylation in the presence of radiolabeled atp and cacl <dig> .
notably, cpk <dig> was auto-phosphorylated along with increased concentration of ca2+, whereas cpk <dig> was not, indicating that cpk <dig> was strictly ca2+-dependent.
auto-phosphorylation of cpk stringently reflects the intensity of the phosphorylation of substrate targets  <cit> .
as shown in figure 7b and additional file  <dig>  cpk <dig> phosphorylated three transcription factors  .
atl <dig>  a member of a multigene family of highly related ring-h <dig> zinc finger proteins that function as e <dig> ubiquitin ligases  <cit>  and a potent regulator of pdf <dig>  transcription  <cit> , was also phosphorylated by cpk <dig> .
addition of bapta, a calcium chelator, to the reaction mixture abolished the phosphorylation by cpk <dig>  suggesting that these phosphorylations were strictly ca2+-dependent.
this result was in line with the ca2+-dependent auto-phosphorylation of cpk <dig>  described above.
in contrast, the cpk13-derived protein labeling  was scarcely activated by the addition of ca2+ ion , suggesting that cpk <dig> has very high sensitivity for autophosphorylation, like soybean cdpkα  <cit> .
cpk <dig> strongly phosphorylated hsfb2a, irrespective of the presence of bapta.
although it has been claimed that myc <dig>  is also involved in pdf <dig>  regulation  <cit> , this transcription factor was not phosphorylated by either cpk <dig> or cpk <dig> .
hsfb2a takes part in the regulation of the herbivore-induced transcription of pdf <dig>
we investigated the pdf <dig>  expression levels in s. littoralis-damaged leaves of arabidopsis wt and hsfb2a t-dna insertion mutants  .
compared to s. littoralis-damaged wt leaves, hsfb2a plants showed significantly lower transcript levels in s. littoralis-damaged leaves.
thus, hsfb2a appeared to be a positive regulator of herbivore-induced pdf <dig>  expression.
in addition, to investigate the in vivo function of cpks, a constitutively active form of cpk and hsfb2a were co-expressed as cofactors for the transient expression of a reporter  gene under the control of the pdf <dig>  promoter in nicotiana benthamiana leaves, in agrobacterium tumefaciens-mediated transient expression  assays.
for these assays, we prepared a constitutively active form of cpk proteins which lacks junction and calmodulin-like domains and thus no longer shows ca2+ dependency.
as shown in figure 8b, the reporter gene activity was drastically increased when co-expressed with hsfb2a as effector.
the activation by hsfb2a was further enhanced  when cpk <dig> and cpk <dig> were co-expressed, respectively.
however, when cpk <dig> or cpk <dig> was co-expressed in the absence of hsfb2a, scarcely any increase of the expression of the reporter gene was observed.
similarly, other cpk substrate transcription factors  were co-expressed as effectors, but neither of them resulted in significant transactivation of the gus marker gene .
discussion
regulation of ca2+ homeostasis is important, particularly when ca2+ is involved as a signaling ion.
in plant cells, ca2+-binding proteins also serve as regulators of internal free ca2+ levels.
protein phosphorylation is the most common type of post-translational modification, and functions through phosphorylation-induced conformational changes  <cit> .
since cpks may be involved in the specificity and cross-talk of signal transduction for a variety of biotic and abiotic stresses, their possible involvement in active signaling cascades in herbivore responses needs to be investigated  <cit> .
the present study provides a new view of a signaling network for plant-insect interactions.
this cascade is not involved in the phytohormone -related signaling pathways, but rather is able to directly impact transcription factors for defense responses.
in fact, we did not observe striking effects of loss of cpk function on the biomass of s. littoralis larvae by feeding cpk <dig> or cpk <dig> mutants compared to wt plants for up to  <dig> days .
this suggests that more genes than just pdf <dig>  contribute in a complex manner to the onset of acquired resistance to the generalist herbivore s. littoralis.
for instance, compared to the levels in wt leaves infested by s. littoralis, the leaf transcript levels of thi <dig>  were higher in cpk <dig> after  <dig> h but lower after  <dig> h .
herbivory responses in arabidopsis may not be mediated strikingly by the aba signaling network .
in guard cells, cpk <dig> appears to act in the phosphorylation of plasma membrane s-type anion channels for the ca2+-reactive stomatal closure response  <cit> .
our data show that a loss of cpk <dig> function may consequently lead to an increase of cytosolic ca2+ concentration in the infested leaf cells .
if we consider that aba activation of plasma membrane ca2+-permeable channels is, in contrast, impaired in cpk <dig> or double cpk3cpk <dig> mutant guard cells  <cit> , we can argue that mesophyll cells, which are the most responsive to herbivore attack, respond in a different way compared to guard cells.
in summary, the present findings are consistent with a model in which additional signaling branches function in the herbivory signal transduction network in parallel to cpk3-imposed feedback regulation of ca2+ channels, and these additional branches are different from those of the stomatal closure response controlled via aba signaling.
in contrast to cpk <dig>  cpk <dig> does not act upstream of herbivore-stimulated ca2+ transients .
screening of the databases for cis-acting regulatory dna elements revealed the presence of a gcc box gccgcc  <cit> ) in the pdf <dig>  promoter that is potentially recognized by erf <dig>  which is one of the cpk <dig> substrates  and a potent regulator of pdf <dig>   <cit> .
however, our agroinfiltration assays showed that erf <dig> as a cofactor failed to transactivate the reporter gus gene under the control of the gcc consensus sequence  and the pdf <dig>  promoter in n. benthamiana .
we therefore infer that erf <dig> may interact only indirectly with the pdf <dig>  promoter, similarly to myc <dig>  <cit> .
otherwise, additional cis/trans-factors or protein modifications might be required to fully facilitate this hetero planta system.
according to our agroinfiltration assays, cpk3-or cpk13-derived phosphorylation of a heat shock factor  appeared to be directly and positively involved in pdf <dig>  transcriptional regulation .
heat shock factors  are well known to function in the regulation of stress-inducible genes  by recognizing a conserved binding motif .
however, the pdf <dig>  promoter lacks intact hse motifs.
it is therefore considered that hsfb2a can act as part of the transcriptional machinery of pdf <dig>  transcription by directly binding to non-hse sequence present in the promoter, and then cpk <dig> and cpk <dig> phosphorylate hsfb2a to modulate its activity.
similarly, kumar et al.
<cit>  reported that double knockout hsfb1/hsfb2b plants showed up-regulation of the basal mrna-levels of pdf <dig>  in mutant plants.
therefore, it will be of great interest to identify a novel non-hse dna recognition site for the class b-hsf transcription factors.
hsfb2a belongs to the hsf class b transcription factors .
however, the function of class b-hsfs differs from that of class a-hsfs due to a structural variation within the oligomerization domain and the lack of an aha-motif, which is required for the transcriptional activation function of class a-hsfs  <cit> .
since b-hsfs have the capacity to bind to similar or the same hse sites in the heat shock gene promoters as class a-hsfs, most of them may act as repressors of target gene expression  <cit> .
if class b-hsfs generally antagonistically interact with a-hsfs by binding  to the hse consensus sequence, their regulatory mechanisms would in most cases be different from the positive regulation of the herbivore-induced pdf <dig>  promoter lacking intact hse motifs.
a-hsfs should be investigated as possible additional cofactors in further studies.
CONCLUSIONS
these results reveal the involvement of two arabidopsis cpks  in the herbivory-induced signaling network via hsfb2a-mediated regulation of the defense-related transcriptional machinery.
to reveal whether protein phosphorylation has significant effects on the transcript levels in response to feeding by caterpillars, future studies such as genetic analyses investigating double mutants defective for both cpks and substrates or plants overexpressing those genes will be needed.

 one of the significant factors determining successful plant growth and reproduction is an efficient defense against insect attacks. after herbivore feeding there is a dramatic ca2+ influx limited to a few cell layers lining the damage zone. signals induced rapidly by herbivore attack have been found to spread over the leaf, leading to a strong ca2+-dependent transmembrane potential depolarization in the damage zone followed by a transient vm hyperpolarization in the close vicinity and a constant depolarization at distances greater than 6- mm. these initial cues are transmitted within the plant by signal transduction pathways that include phosphorylation cascades, such as mitogen-activated protein kinases, and the jasmonic acid pathway, which play a central and conserved role in promoting resistance to a broad spectrum of insects. however, there is a missing link to downstream signaling and gene regulation for defense responses. in this network, ca2+-binding sensory proteins are of particular interest, since following ca2+ influx the sensory proteins may secondarily decode information contained in the temporal and spatial patterns of the signal trafficking to control metabolism and gene expression. plants possess several classes of ca2+-binding sensory proteins, including calmodulins, calmodulin-like proteins, calcineurin b-like proteins, and ca2+-dependent protein kinases. the cpks are of special interest, since they represent a novel class of ca2+ sensors, having both a protein kinase domain and a calmodulin-like domain in a single polypeptide. cpks constitute a large family of serine/threonine protein kinases that are broadly distributed in the plant kingdom. for example, the arabidopsis genome is predicted to have different cpks. arabidopsis cpk was the first cpk to be characterized, and is known to be activated by phospholipids and 14-3- proteins, which are small, highly conserved eukaryotic proteins that regulate multiple cellular enzymes, including protein kinases. atcpks and act as abscisic acid signaling components, and are involved in aba-responsive gene expression, seed germination, seedling growth, and stomatal movement. especially, atcpks and are likely to interact with aba-related leucine zipper class transcription factors, indicating the proximate involvement of cpks in transcriptional regulation. curiously, little attention has been given to the role of cpks in defense responses. only one case has been reported: in tobacco, ntcdpk modulates the activation of stress-induced map kinases, and this interaction requires the synthesis and perception of wound hormones. the role of cpks involved in the defense response against insect herbivory has never been reported. in this report, we show the involvement of two arabidopsis cpks in the herbivory-induced signaling network via post-translational regulation of the defense-related transcriptional machinery. implications for possible signal trafficking via cpks are discussed.  cpk and cpk mutants showed decreased transcript levels of defense genes in s. littoralis-damaged leaves we obtained t-dna insertion mutant lines that were putative atcpk-deficient mutants from the european arabidopsis stock centre. homozygous t-dna insertion lines corresponding to each cpk gene were confirmed using the pcr method and challenged with herbivore damage. transcript levels of plant defensin gene pdf, which is induced in wild-type leaves exposed to spodoptera littoralis larvae for h, were investigated in the mutant leaves. compared to s. littoralis-damaged wt leaves, t-dna insertion line cpk and two different cpk lines and cpk13- ) showed significantly lower transcript levels in s. littoralis-damaged leaves. the molecular analysis of cpk t-dna insertion mutants and gene expression profiling of the mutants are shown in additional file in addition to our study of pdf, the time-courses of the transcript levels of two other defense-related genes after herbivore attack were examined in cpk cpk13- and cpk13- leaves and compared to those in wt leaves. the transcript level of thi in wt leaves was increased about fold after h but declined after h. the cpk mutant showed a comparable level of the thi expression during the time course, whereas the transcript levels in cpk13- and cpk13- leaves remained unchanged at h, but increased after h. thus, wt and cpk probably have different temporal regulation. throughout the time-course, the induced transcript levels of vsp were comparable between the wt and all the mutants. possible involvement of phytohormone signaling in the herbivory-related cpk cascades to assess whether loss of cpk function affects the signal transduction involved in the defense response, we explored the biosynthesis of ja, jasmonyl-l-isoleucine, ethylene, and abscisic acid. as shown in figure all the phytohormones examined in this study were formed and accumulated at a similar rate in the infested wt, compared to the cpk mutant leaves, indicating that cpk and cpk are not upstream signal kinases for the biosynthesis of ja, aba and ethylene. we also investigated the pdf expression levels in wt and in cpk and cpk mutants by applying an exogenous solution of either ja, ethephon, or aba. compared to the control, after h the transcript levels of pdf were very slightly induced by ja or aba to levels comparable to those in wt and mutant leaves. ethephon treatment resulted in drastic but comparable increases of the transcript in both wt and mutant leaves. we therefore conclude that the cpk and cpk cascades are not closely linked to the above phytohormone signaling and biosynthesis cascades. increased intracellular ca2+ levels in cpk leaf cells after herbivore and mechanical damage it is interesting to note that the cpk mutant showed abnormal changes of the cytosolic ca2+ level after insect damage. as shown in figure when the membrane-permeable fluo- am was applied to wt leaf tissues, it showed a cytoplasmic subcellular localization at sites damaged by s. littoralis or after mechanical wounding. cpk but not cpk13- plants showed a more marked increase of the cytosolic ca2+ level after the damage, compared to wt. substrate targeting of cpk and cpk13 in previous studies, cpk was found to be localized in the nucleus and the cytosol. in this study, we additionally tested the subcellular localization of cpk13-gfp fusion proteins in transiently expressing onion peels, which revealed nuclear, cytosolic and plasma membrane localizations of the fusion proteins. accordingly, in order to screen the protein target of cpk and cpk protein kinase assays with nuclear and cytosolic protein substrates synthesized using the wheat germ cell-free system were carried out. cpk or cpk proteins purified from a cell-free extract were incubated with radiolabeled atp, cacl and the distinct substrates. both cpk and cpk showed auto-phosphorylation in the presence of radiolabeled atp and cacl. notably, cpk was auto-phosphorylated along with increased concentration of ca2+, whereas cpk was not, indicating that cpk was strictly ca2+-dependent. auto-phosphorylation of cpk stringently reflects the intensity of the phosphorylation of substrate targets. as shown in figure 7b and additional file cpk phosphorylated three transcription factors. atl a member of a multigene family of highly related ring-h zinc finger proteins that function as e ubiquitin ligases and a potent regulator of pdf transcription, was also phosphorylated by cpk. addition of bapta, a calcium chelator, to the reaction mixture abolished the phosphorylation by cpk suggesting that these phosphorylations were strictly ca2+-dependent. this result was in line with the ca2+-dependent auto-phosphorylation of cpk described above. in contrast, the cpk13-derived protein labeling was scarcely activated by the addition of ca2+ ion, suggesting that cpk has very high sensitivity for autophosphorylation, like soybean cdpkα. cpk strongly phosphorylated hsfb2a, irrespective of the presence of bapta. although it has been claimed that myc is also involved in pdf regulation, this transcription factor was not phosphorylated by either cpk or cpk. hsfb2a takes part in the regulation of the herbivore-induced transcription of pdf we investigated the pdf expression levels in s. littoralis-damaged leaves of arabidopsis wt and hsfb2a t-dna insertion mutants. compared to s. littoralis-damaged wt leaves, hsfb2a plants showed significantly lower transcript levels in s. littoralis-damaged leaves. thus, hsfb2a appeared to be a positive regulator of herbivore-induced pdf expression. in addition, to investigate the in vivo function of cpks, a constitutively active form of cpk and hsfb2a were co-expressed as cofactors for the transient expression of a reporter gene under the control of the pdf promoter in nicotiana benthamiana leaves, in agrobacterium tumefaciens-mediated transient expression assays. for these assays, we prepared a constitutively active form of cpk proteins which lacks junction and calmodulin-like domains and thus no longer shows ca2+ dependency. as shown in figure 8b, the reporter gene activity was drastically increased when co-expressed with hsfb2a as effector. the activation by hsfb2a was further enhanced when cpk and cpk were co-expressed, respectively. however, when cpk or cpk was co-expressed in the absence of hsfb2a, scarcely any increase of the expression of the reporter gene was observed. similarly, other cpk substrate transcription factors were co-expressed as effectors, but neither of them resulted in significant transactivation of the gus marker gene. discussion regulation of ca2+ homeostasis is important, particularly when ca2+ is involved as a signaling ion. in plant cells, ca2+-binding proteins also serve as regulators of internal free ca2+ levels. protein phosphorylation is the most common type of post-translational modification, and functions through phosphorylation-induced conformational changes. since cpks may be involved in the specificity and cross-talk of signal transduction for a variety of biotic and abiotic stresses, their possible involvement in active signaling cascades in herbivore responses needs to be investigated. the present study provides a new view of a signaling network for plant-insect interactions. this cascade is not involved in the phytohormone -related signaling pathways, but rather is able to directly impact transcription factors for defense responses. in fact, we did not observe striking effects of loss of cpk function on the biomass of s. littoralis larvae by feeding cpk or cpk mutants compared to wt plants for up to days. this suggests that more genes than just pdf contribute in a complex manner to the onset of acquired resistance to the generalist herbivore s. littoralis. for instance, compared to the levels in wt leaves infested by s. littoralis, the leaf transcript levels of thi were higher in cpk after h but lower after h. herbivory responses in arabidopsis may not be mediated strikingly by the aba signaling network. in guard cells, cpk appears to act in the phosphorylation of plasma membrane s-type anion channels for the ca2+-reactive stomatal closure response. our data show that a loss of cpk function may consequently lead to an increase of cytosolic ca2+ concentration in the infested leaf cells. if we consider that aba activation of plasma membrane ca2+-permeable channels is, in contrast, impaired in cpk or double cpk3cpk mutant guard cells, we can argue that mesophyll cells, which are the most responsive to herbivore attack, respond in a different way compared to guard cells. in summary, the present findings are consistent with a model in which additional signaling branches function in the herbivory signal transduction network in parallel to cpk3-imposed feedback regulation of ca2+ channels, and these additional branches are different from those of the stomatal closure response controlled via aba signaling. in contrast to cpk cpk does not act upstream of herbivore-stimulated ca2+ transients. screening of the databases for cis-acting regulatory dna elements revealed the presence of a gcc box gccgcc ) in the pdf promoter that is potentially recognized by erf which is one of the cpk substrates and a potent regulator of pdf. however, our agroinfiltration assays showed that erf as a cofactor failed to transactivate the reporter gus gene under the control of the gcc consensus sequence and the pdf promoter in n. benthamiana. we therefore infer that erf may interact only indirectly with the pdf promoter, similarly to myc. otherwise, additional cis/trans-factors or protein modifications might be required to fully facilitate this hetero planta system. according to our agroinfiltration assays, cpk3-or cpk13-derived phosphorylation of a heat shock factor appeared to be directly and positively involved in pdf transcriptional regulation. heat shock factors are well known to function in the regulation of stress-inducible genes by recognizing a conserved binding motif. however, the pdf promoter lacks intact hse motifs. it is therefore considered that hsfb2a can act as part of the transcriptional machinery of pdf transcription by directly binding to non-hse sequence present in the promoter, and then cpk and cpk phosphorylate hsfb2a to modulate its activity. similarly, kumar et al. reported that double knockout hsfb1/hsfb2b plants showed up-regulation of the basal mrna-levels of pdf in mutant plants. therefore, it will be of great interest to identify a novel non-hse dna recognition site for the class b-hsf transcription factors. hsfb2a belongs to the hsf class b transcription factors. however, the function of class b-hsfs differs from that of class a-hsfs due to a structural variation within the oligomerization domain and the lack of an aha-motif, which is required for the transcriptional activation function of class a-hsfs. since b-hsfs have the capacity to bind to similar or the same hse sites in the heat shock gene promoters as class a-hsfs, most of them may act as repressors of target gene expression. if class b-hsfs generally antagonistically interact with a-hsfs by binding to the hse consensus sequence, their regulatory mechanisms would in most cases be different from the positive regulation of the herbivore-induced pdf promoter lacking intact hse motifs. a-hsfs should be investigated as possible additional cofactors in further studies.  these results reveal the involvement of two arabidopsis cpks in the herbivory-induced signaling network via hsfb2a-mediated regulation of the defense-related transcriptional machinery. to reveal whether protein phosphorylation has significant effects on the transcript levels in response to feeding by caterpillars, future studies such as genetic analyses investigating double mutants defective for both cpks and substrates or plants overexpressing those genes will be needed. 
furthermore, in vivo agroinfiltration assays showed that cpk3-or cpk13-derived phosphorylation of a heat shock factor promotes pdf transcriptional activation in the defense response. this cascade is not involved in the phytohormone-related signaling pathways, but rather directly impacts transcription factors for defense responses. these results reveal the involvement of two arabidopsis cpks in the herbivory-induced signaling network via hsfb2a-mediated regulation of the defense-related transcriptional machinery. ca2+-binding sensory proteins such as ca2+-dependent protein kinases have been predicted to mediate the signaling following ca2+ influx after insect herbivory. following insect attack, the cpk and cpk mutants showed lower transcript levels of plant defensin gene pdf compared to wild-type plants.
 plant ca2+ signals are involved in a wide array of intracellular signaling pathways after pest invasion. ca2+-binding sensory proteins such as ca2+-dependent protein kinases have been predicted to mediate the signaling following ca2+ influx after insect herbivory. however, until now this prediction was not testable.  to investigate the roles cpks play in a herbivore response-signaling pathway, we screened the characteristics of arabidopsis cpk mutants damaged by a feeding generalist herbivore, spodoptera littoralis. following insect attack, the cpk and cpk mutants showed lower transcript levels of plant defensin gene pdf compared to wild-type plants. the cpk cascade was not directly linked to the herbivory-induced signaling pathways that were mediated by defense-related phytohormones such as jasmonic acid and ethylene. cpk was also suggested to be involved in a negative feedback regulation of the cytosolic ca2+ levels after herbivory and wounding damage. in vitro kinase assays of cpk protein with a suite of substrates demonstrated that the protein phosphorylates transcription factors in the presence of ca2+. cpk strongly phosphorylated only hsfb2a, irrespective of the presence of ca2+. furthermore, in vivo agroinfiltration assays showed that cpk3-or cpk13-derived phosphorylation of a heat shock factor promotes pdf transcriptional activation in the defense response.  these results reveal the involvement of two arabidopsis cpks in the herbivory-induced signaling network via hsfb2a-mediated regulation of the defense-related transcriptional machinery. this cascade is not involved in the phytohormone-related signaling pathways, but rather directly impacts transcription factors for defense responses. 