Calcium-dependent protein kinases （CDPKs） comprise a family of plant serine/threonine protein kinases in which the calcium sensing domain and the kinase effector domain are combined within one molecule. So far, a bi...Calcium-dependent protein kinases （CDPKs） comprise a family of plant serine/threonine protein kinases in which the calcium sensing domain and the kinase effector domain are combined within one molecule. So far, a biological function in abiotic stress signaling has only been reported for few CDPK isoforms, whereas the underlying biochemical mechanism for these CDPKs is still mainly unknown. Here, we show that CPK21 from Arabidopsis thaliana is biochemically activated in vivo in response to hyperosmotic stress. Loss-of-function seedlings of cpk21 are more tolerant to hyperosmotic stress and mutant plants show increased stress responses with respect to marker gene expression and metabolite accumulation. In transgenic Arabidopsis complementation lines in the cpk21 mutant background, in which either CPK21 wildtype, or a full-length enzyme variant carrying an amino-acid substitution were stably expressed, stress responsitivity was restored by CPK21 but not with the kinase inactive variant. The biochemical characterization of in planta synthesized and purified CPK21 protein revealed that within the calcium-binding domain, N-terminal EF1- and EF2-motifs compared to C-terminal EF3- and EF4-motifs differ in their contribution to calcium-regulated kinase activity, suggesting a crucial role for the N-terminal EF-hand pair. Our data provide evidence for CPK21 contributing in abiotic stress signaling and suggest that the N-terminal EF-hand pair is a calcium-sensing determinant controlling specificity of CPK21 function.展开更多
Patch clamp techniques were employed to investigate if calcium dependent protein kinases (CDPKs) be involved in the signal transduction pathways of stomatal movement regulation by the phytohormone abscisic acid (ABA...Patch clamp techniques were employed to investigate if calcium dependent protein kinases (CDPKs) be involved in the signal transduction pathways of stomatal movement regulation by the phytohormone abscisic acid (ABA) in Vicia faba. Stomatal opening was completely inhibited by external application of 1 μmol/L ABA, and such ABA inhibition was significantly reversed by the addition of CDPK inhibitor trifluoperazine (TFP). The inward whole cell K + currents were inhibited by 60% in the presence of 1 μmol/L intracellular ABA, and this inhibition was completely abolished by the addition of CDPK competitive substrate histone Ⅲ S. The results suggest that CDPKs may be involved in the signal transduction cascades of ABA regulated stomatal movements.展开更多
Calcium-dependent protein kinases （CDPKs, EC 2.7.1.37） comprise a large family of Ser/Thr kinases in plants and play an important role in plant Ca^2＋ signal transduction. A full-length CDPK gene, NtCDPK12 （GenBank...Calcium-dependent protein kinases （CDPKs, EC 2.7.1.37） comprise a large family of Ser/Thr kinases in plants and play an important role in plant Ca^2＋ signal transduction. A full-length CDPK gene, NtCDPK12 （GenBank accession number GQ337420）, was isolated from common tobacco （Nicotiana tabacum） leaves by rapid amplification of eDNA ends （RACE）. The NtCDPK12 eDNA is 1 816 bp length and contains an open reading frame （ORF） of 1 461 bp encoding 486 amino acids. Sequence alignments indicated that NtCDPK12 contains all conserved regions found in CDPKs and shows a high level of sequence similarity to many other plant CDPKs. The results of real-time quantitative reverse transcription-PCR （qRT- PCR） showed that NtCDPK12 was highly expressed in stems and increased in roots treated with high-salt or subjected to drought stress, which indicates that NtCDPK12 was induced by high-salt and drought stresses.展开更多
为给进一步分离和克隆植物中的CDPK基因奠定基础,以已经鉴定的拟南芥的CDPK(Calcium-dependent protein kinases)基因编码的结构域为检索序列,在基因组水平上对杨树的CDPK基因家族的成员进行分析,结果共找到36个杨树CDPK基因。同时利用...为给进一步分离和克隆植物中的CDPK基因奠定基础,以已经鉴定的拟南芥的CDPK(Calcium-dependent protein kinases)基因编码的结构域为检索序列,在基因组水平上对杨树的CDPK基因家族的成员进行分析,结果共找到36个杨树CDPK基因。同时利用这些基因编码的蛋白质序列与拟南芥中的CDPK基因构建了系统进化树,并对这些蛋白序列的保守序列进行了分析。结果表明,杨树CDPK基因内含子的插入位置相对比较保守。与拟南芥相比,杨树CDPK基因家族成员含有更多的基因对。通过对拟南芥、杨树CDPK家族之间进化关系和内含子分布比较发现,一些CDPK家族成员在拟南芥和杨树未分离之前就已经形成。展开更多
As calcium sensors, plant calcium-dependent protein kinases(CDPKs) play important roles in plants' responses to various abiotic stresses. Here, we report the functional characterization of CPK28, a member of the C...As calcium sensors, plant calcium-dependent protein kinases(CDPKs) play important roles in plants' responses to various abiotic stresses. Here, we report the functional characterization of CPK28, a member of the CDPK family in Arabidopsis, in response to osmotic stress.The cpk28 mutant, a loss-of-function mutant, exhibited an NaCl- and mannitol-sensitive phenotype in green cotyledons, while CPK28-overexpressing plants displayed stronger tolerance to NaCl and mannitol stresses than wildtype plants. Reverse transcriptase-polymerase chain reaction and beta-glucuronidase staining assays showed that NaCl and mannitol stresses induced CPK28. CPK28-overexpressing lines accumulated significantly more proline relative to wild-type plants and mutant plants under NaCl and mannitol stresses. Transient expression of CPK28-GFP in mesophyll cell protoplasts, as well as stable transgenic lines expressing CPK28-GFP, showed that CPK28 was localized in the plasma membrane. Expression levels of known stress-responsive genes were not significantly altered in the null mutant and overexpression lines,suggesting that CPK28 possibly mediated the stress response via the regulation of target proteins rather than via regulation at the level of transcription. Meanwhile, CPK28could autophosphorylate. Taken together, these data demonstrated that CPK28, a potential positive regulator, is involved in the response to osmotic stress in Arabidopsis.展开更多
Calcium oscillations are induced by different stresses.Calcium-dependent protein kinases(CDPKs/CPKs)are one major group of the plant calcium decoders that are involved in various processes including drought response.S...Calcium oscillations are induced by different stresses.Calcium-dependent protein kinases(CDPKs/CPKs)are one major group of the plant calcium decoders that are involved in various processes including drought response.Some CPKs are calcium-independent.Here,we identified ZmCPK2 as a negative regulator of drought resistance by screening an overexpression transgenic maize pool.We found that ZmCPK2 does not bind calcium,and its activity is mainly inhibited during short term abscisic acid(ABA)treatment,and dynamically changed in prolonged treatment.Interestingly,ZmCPK2 interacts with and is inhibited by calcium-dependent ZmCPK17,a positive regulator of drought resistance,which is activated by ABA.ZmCPK17 could prevent the nuclear localization of ZmCPK2 through phosphorylation of ZmCPK2T60.ZmCPK2 interacts with and phosphorylates and activates ZmYAB15,a negative transcriptional factor for drought resistance.Our results suggest that drought stress-induced Ca^(2+)can be decoded directly by ZmCPK17 that inhibits ZmCPK2,thereby promoting plant adaptation to water deficit.展开更多
文摘Calcium-dependent protein kinases （CDPKs） comprise a family of plant serine/threonine protein kinases in which the calcium sensing domain and the kinase effector domain are combined within one molecule. So far, a biological function in abiotic stress signaling has only been reported for few CDPK isoforms, whereas the underlying biochemical mechanism for these CDPKs is still mainly unknown. Here, we show that CPK21 from Arabidopsis thaliana is biochemically activated in vivo in response to hyperosmotic stress. Loss-of-function seedlings of cpk21 are more tolerant to hyperosmotic stress and mutant plants show increased stress responses with respect to marker gene expression and metabolite accumulation. In transgenic Arabidopsis complementation lines in the cpk21 mutant background, in which either CPK21 wildtype, or a full-length enzyme variant carrying an amino-acid substitution were stably expressed, stress responsitivity was restored by CPK21 but not with the kinase inactive variant. The biochemical characterization of in planta synthesized and purified CPK21 protein revealed that within the calcium-binding domain, N-terminal EF1- and EF2-motifs compared to C-terminal EF3- and EF4-motifs differ in their contribution to calcium-regulated kinase activity, suggesting a crucial role for the N-terminal EF-hand pair. Our data provide evidence for CPK21 contributing in abiotic stress signaling and suggest that the N-terminal EF-hand pair is a calcium-sensing determinant controlling specificity of CPK21 function.
文摘Patch clamp techniques were employed to investigate if calcium dependent protein kinases (CDPKs) be involved in the signal transduction pathways of stomatal movement regulation by the phytohormone abscisic acid (ABA) in Vicia faba. Stomatal opening was completely inhibited by external application of 1 μmol/L ABA, and such ABA inhibition was significantly reversed by the addition of CDPK inhibitor trifluoperazine (TFP). The inward whole cell K + currents were inhibited by 60% in the presence of 1 μmol/L intracellular ABA, and this inhibition was completely abolished by the addition of CDPK competitive substrate histone Ⅲ S. The results suggest that CDPKs may be involved in the signal transduction cascades of ABA regulated stomatal movements.
基金supported in part by the Special Grand Science and Technology Projects for China National Tobacco Corporation (110200701022,110200902036),Chinathe open subject from the State Key Laboratory of Plant Physiology and Biochemistry (PPB08004)
文摘Calcium-dependent protein kinases （CDPKs, EC 2.7.1.37） comprise a large family of Ser/Thr kinases in plants and play an important role in plant Ca^2＋ signal transduction. A full-length CDPK gene, NtCDPK12 （GenBank accession number GQ337420）, was isolated from common tobacco （Nicotiana tabacum） leaves by rapid amplification of eDNA ends （RACE）. The NtCDPK12 eDNA is 1 816 bp length and contains an open reading frame （ORF） of 1 461 bp encoding 486 amino acids. Sequence alignments indicated that NtCDPK12 contains all conserved regions found in CDPKs and shows a high level of sequence similarity to many other plant CDPKs. The results of real-time quantitative reverse transcription-PCR （qRT- PCR） showed that NtCDPK12 was highly expressed in stems and increased in roots treated with high-salt or subjected to drought stress, which indicates that NtCDPK12 was induced by high-salt and drought stresses.
文摘为给进一步分离和克隆植物中的CDPK基因奠定基础,以已经鉴定的拟南芥的CDPK(Calcium-dependent protein kinases)基因编码的结构域为检索序列,在基因组水平上对杨树的CDPK基因家族的成员进行分析,结果共找到36个杨树CDPK基因。同时利用这些基因编码的蛋白质序列与拟南芥中的CDPK基因构建了系统进化树,并对这些蛋白序列的保守序列进行了分析。结果表明,杨树CDPK基因内含子的插入位置相对比较保守。与拟南芥相比,杨树CDPK基因家族成员含有更多的基因对。通过对拟南芥、杨树CDPK家族之间进化关系和内含子分布比较发现,一些CDPK家族成员在拟南芥和杨树未分离之前就已经形成。
基金supported by the National Genetically Modified Organisms Breeding Major Projects(2011ZX08009-002)
文摘As calcium sensors, plant calcium-dependent protein kinases(CDPKs) play important roles in plants' responses to various abiotic stresses. Here, we report the functional characterization of CPK28, a member of the CDPK family in Arabidopsis, in response to osmotic stress.The cpk28 mutant, a loss-of-function mutant, exhibited an NaCl- and mannitol-sensitive phenotype in green cotyledons, while CPK28-overexpressing plants displayed stronger tolerance to NaCl and mannitol stresses than wildtype plants. Reverse transcriptase-polymerase chain reaction and beta-glucuronidase staining assays showed that NaCl and mannitol stresses induced CPK28. CPK28-overexpressing lines accumulated significantly more proline relative to wild-type plants and mutant plants under NaCl and mannitol stresses. Transient expression of CPK28-GFP in mesophyll cell protoplasts, as well as stable transgenic lines expressing CPK28-GFP, showed that CPK28 was localized in the plasma membrane. Expression levels of known stress-responsive genes were not significantly altered in the null mutant and overexpression lines,suggesting that CPK28 possibly mediated the stress response via the regulation of target proteins rather than via regulation at the level of transcription. Meanwhile, CPK28could autophosphorylate. Taken together, these data demonstrated that CPK28, a potential positive regulator, is involved in the response to osmotic stress in Arabidopsis.
基金supported by grants from the National Science Foundation of China(32030008 and 31921001)the Beijing Outstanding University Discipline(2022YFF1001600).
文摘Calcium oscillations are induced by different stresses.Calcium-dependent protein kinases(CDPKs/CPKs)are one major group of the plant calcium decoders that are involved in various processes including drought response.Some CPKs are calcium-independent.Here,we identified ZmCPK2 as a negative regulator of drought resistance by screening an overexpression transgenic maize pool.We found that ZmCPK2 does not bind calcium,and its activity is mainly inhibited during short term abscisic acid(ABA)treatment,and dynamically changed in prolonged treatment.Interestingly,ZmCPK2 interacts with and is inhibited by calcium-dependent ZmCPK17,a positive regulator of drought resistance,which is activated by ABA.ZmCPK17 could prevent the nuclear localization of ZmCPK2 through phosphorylation of ZmCPK2T60.ZmCPK2 interacts with and phosphorylates and activates ZmYAB15,a negative transcriptional factor for drought resistance.Our results suggest that drought stress-induced Ca^(2+)can be decoded directly by ZmCPK17 that inhibits ZmCPK2,thereby promoting plant adaptation to water deficit.
