Search

Background: Zinc finger proteins (ZFPs) are important regulators in abiotic and biotic stress tolerance in plants. However, the role of the ZFPs in wheat responding to pathogen infection is poorly understood. Results: In this study, we found TaZFP8-5B was down-regulated by Puccinia striiformis f. sp. tritici (Pst) infection. TaZFP8-5B possesses a single C2H2-type zinc finger domain with a plant-specific QALGGH motif, and an EAR motif (LxLxL) at the C-terminus. The EAR motif represses the trans-activation ability of TaZFP8-5B. Knocking down the expression of TaZFP8 by virus-induced gene silencing increased wheat resistance to Pst, whereas TaZFP8-5B-overexpressing reduced wheat resistance to stripe rust and rice resistance to Magnaporthe oryzae, suggesting that TaZFP8-5B plays a negative role in the modulation of plant immunity. Using bimolecular fluorescence complementation, split-luciferase, and yeast two-hybrid assays, we showed that TaZFP8-5B interacted with a wheat calmodulin-like protein TaCML21. Knock-down of TaCML21 reduced wheat resistance to Pst. Conclusions: This study characterized the function of TaZFP8-5B and its interacting protein TaCML21. Our findings provide a new perspective on a regulatory module made up of TaCML21-TaZFP8-5B in plant immunity. [ABSTRACT FROM AUTHOR]

Calmodulins (CaMs) and calmodulin-like proteins (CMLs) belong to families of calcium-sensors that act as calcium ion (Ca2+) signal-decoding proteins and regulate downstream target proteins. As a tropical halophyte, Canavalia rosea shows great resistance to multiple abiotic stresses, including high salinity/alkalinity, extreme drought, heat, and intense sunlight. However, investigations of calcium ion signal transduction involved in the stress responses of C. rosea are limited. The CaM and CML gene families have been identified and characterized in many other plant species. Nevertheless, there is limited available information about these genes in C. rosea. In this study, a bioinformatic analysis, including the gene structures, conserved protein domains, phylogenetic relationships, chromosome distribution, and gene synteny, was comprehensively performed to identify and characterize CrCaMs and CrCMLs. A spatio-temporal expression assay in different organs and environmental conditions was then conducted using the RNA sequencing technique. Additionally, several CrCaM and CrCML members were then cloned and functionally characterized using the yeast heterogeneous expression system, and some of them were found to change the tolerance of yeast to heat, salt, alkalinity, and high osmotic stresses. The results of this study provide a foundation for understanding the possible roles of the CrCaM and CrCML genes, especially for halophyte C. rosea's natural ecological adaptability for its native habitats. This study also provides a theoretical basis for further study of the physiological and biochemical functions of plant CaMs and CMLs that are involved in tolerance to multiple abiotic stresses. [ABSTRACT FROM AUTHOR]

Plant calmodulins (CaMs) and calmodulin-like proteins (CMLs) mediate Ca2+ signaling in response to abiotic stresses. Manipulation of this signaling in crops could increase stress tolerance. We review methods for detecting Ca2+ signals, regulatory roles of CaMs and CMLs, binding targets, and Ca2+ networks under abiotic stress in organelles. [ABSTRACT FROM AUTHOR]

Abstract Background Zinc finger proteins (ZFPs) are important regulators in abiotic and biotic stress tolerance in plants. However, the role of the ZFPs in wheat responding to pathogen infection is poorly understood. Results In this study, we found TaZFP8-5B was down-regulated by Puccinia striiformis f. sp. tritici (Pst) infection. TaZFP8-5B possesses a single C2H2-type zinc finger domain with a plant-specific QALGGH motif, and an EAR motif (LxLxL) at the C-terminus. The EAR motif represses the trans-activation ability of TaZFP8-5B. Knocking down the expression of TaZFP8 by virus-induced gene silencing increased wheat resistance to Pst, whereas TaZFP8-5B-overexpressing reduced wheat resistance to stripe rust and rice resistance to Magnaporthe oryzae, suggesting that TaZFP8-5B plays a negative role in the modulation of plant immunity. Using bimolecular fluorescence complementation, split-luciferase, and yeast two-hybrid assays, we showed that TaZFP8-5B interacted with a wheat calmodulin-like protein TaCML21. Knock-down of TaCML21 reduced wheat resistance to Pst. Conclusions This study characterized the function of TaZFP8-5B and its interacting protein TaCML21. Our findings provide a new perspective on a regulatory module made up of TaCML21-TaZFP8-5B in plant immunity.

Copyright of Acta Agronomica Sinica is the property of Crop Science Society of China and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Summary: It is well known that calcium, ethylene and abscisic acid (ABA) can regulate fruit ripening, however, their interaction in the regulation of fruit ripening has not yet been fully clarified. The present study found that the expression of the papaya calcium sensor CpCML15 was strongly linked to fruit ripening. CpCML15 could bind Ca2+ and served as a true calcium sensor. CpCML15 interacted with CpPP2C46 and CpPP2C65, the candidate components of the ABA signalling pathways. CpPP2C46/65 expression was also related to fruit ripening and regulated by ethylene. CpCML15 was located in the nucleus and CpPP2C46/65 were located in both the nucleus and membrane. The interaction between CpCML15 and CpPP2C46/65 was calcium dependent and further repressed the activity of CpPP2C46/65 in vitro. The transient overexpression of CpCML15 and CpPP2C46/65 in papaya promoted fruit ripening and gene expression related to ripening. The reduced expression of CpCML15 and CpPP2C46/65 by virus‐induced gene silencing delayed fruit colouring and softening and repressed the expression of genes related to ethylene signalling and softening. Moreover, ectopic overexpression of CpCML15 in tomato fruit also promoted fruit softening and ripening by increasing ethylene production and enhancing gene expression related to ripening. Additionally, CpPP2C46 interacted with CpABI5, and CpPP2C65 interacted with CpERF003‐like, two transcriptional factors in ABA and ethylene signalling pathways that are closely related to fruit ripening. Taken together, our results showed that CpCML15 and CpPP2Cs positively regulated fruit ripening, and their interaction integrated the cross‐talk of calcium, ABA and ethylene signals in fruit ripening through the CpCML15‐CpPP2Cs‐CpABI5/CpERF003‐like pathway. [ABSTRACT FROM AUTHOR]

The root‐knot nematode Meloidogyne graminicola secretes effectors into rice tissues to modulate host immunity. Here, we characterised MgCRT1, a calreticulin protein of M. graminicola, and identified its target in the plant. In situ hybridisation showed MgCRT1 mRNA accumulating in the subventral oesophageal gland in J2 nematodes. Immunolocalization indicated MgCRT1 localises in the giant cells during parasitism. Host‐induced gene silencing of MgCRT1 reduced the infection ability of M. graminicola, while over‐expressing MgCRT1 enhanced rice susceptibility to M. graminicola. A yeast two‐hybrid approach identified the calmodulin‐like protein OsCML31 as an interactor of MgCRT1. OsCML31 interacts with the high mobility group protein OsHMGB1 which is a conserved DNA binding protein. Knockout of OsCML31 or overexpression of OsHMGB1 in rice results in enhanced susceptibility to M. graminicola. In contrast, overexpression of OsCML31 or knockout of OsHMGB1 in rice decreases susceptibility to M. graminicola. The GST‐pulldown and luciferase complementation imaging assay showed that MgCRT1 decreases the interaction of OsCML31 and OsHMGB1 in a competitive manner. In conclusion, when M. graminicola infects rice and secretes MgCRT1 into rice, MgCRT1 interacts with OsCML31 and decreases the association of OsCML31 with OsHMGB1, resulting in the release of OsHMGB1 to enhance rice susceptibility. Summary statement: A plant‐pathogenic nematode effector MgCRT1 interacts with OsCML31 and decreases the association of OsCML31 with OsHMGB1 in a competitive manner, resulting in the release of OsHMGB1 to enhance rice susceptibility. [ABSTRACT FROM AUTHOR]

Summary: Transient and rapid increase in cytosolic Ca2+ plays a crucial role in plant–pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI). Cyclic nucleotide‐gated channels (CNGCs) have been implicated in mediating this Ca2+ influx; however, their regulatory mechanisms remain poorly understood.Here, we have found that AVRblb2 requires the calmodulin (CaM) and calmodulin‐like (CML) proteins as co‐factors to interact with the NbCNGCs, resulting in the formation of AVRblb2‐CaM/CML‐NbCNGCs complex. Furthermore, CaM and CML are dissociated from NbCNGC18 during PTI response to increase Ca2+ influx; however, Avrblb2 inhibits calcium channel activation by disrupting the release of CaM and CML from NbCNGC18.Following recognition of PAMP, NbCNGC18 forms active heteromeric channels with other NbCNGCs, which may give selectivity of CNGC complex against diverse signals for fine‐tuning of cytosolic Ca2+ level to mediate appropriate responses. Silencing of multiple NbCNGCs compromised the function of AVRblb2 on the pathogenicity of Phytophthora infestans, confirming that AVRblb2 contributes to pathogen virulence by targeting CNGCs.Our findings provide new insights into the regulation of CNGCs in PTI and the role of pathogen effectors in manipulating host cell physiology to promote infection. [ABSTRACT FROM AUTHOR]

SUMMARY: Wheat yellow mosaic virus (WYMV) causes severe wheat viral disease in Asia. However, the viral suppressor of RNA silencing (VSR) encoded by WYMV has not been identified. Here, the P1 protein encoded by WYMV RNA2 was shown to suppress RNA silencing in Nicotiana benthamiana. Mutagenesis assays revealed that the alanine substitution mutant G175A of P1 abolished VSR activity and mutant Y10A VSR activity remained only in younger leaves. P1, but not G175A, interacted with gene silencing‐related protein, N. benthamiana calmodulin‐like protein (NbCaM), and calmodulin‐binding transcription activator 3 (NbCAMTA3), and Y10A interacted with NbCAMTA3 only. Competitive Bimolecular fluorescence complementation and co‐immunoprecipitation assays showed that the ability of P1 disturbing the interaction between NbCaM and NbCAMTA3 was stronger than Y10A, Y10A was stronger than G175A. In vitro transcript inoculation of infectious WYMV clones further demonstrated that VSR‐defective mutants G175A and Y10A reduced WYMV infection in wheat (Triticum aestivum L.), G175A had a more significant effect on virus accumulation in upper leaves of wheat than Y10A. Moreover, RNA silencing, temperature, and autophagy have significant effects on the accumulation of P1 in N. benthamiana. Taken together, WYMV P1 acts as VSR by interfering with calmodulin‐associated antiviral RNAi defense to facilitate virus infection in wheat, which has provided clear insights into the function of P1 in the process of WYMV infection. Significance Statement: This study reveals that the wheat yellow mosaic virus (WYMV) P1 acts as VSR by interfering with calmodulin‐associated antiviral RNAi defense and facilitates virus infection in wheat. Results of our research provide clear insights and solutions for further understanding the role of P1 in process of WYMV infection. [ABSTRACT FROM AUTHOR]

Calmodulins (CaMs) and Calmodulin-like proteins (CMLs) are vital in plant growth, development, and stress responses. However, CaMs and CMLs have not been fully identified and characterized in brown algae, which has been evolving independently of the well-studied green plant lineage. In this study, whole-genome searches revealed one SjCaM and eight SjCMLs in Saccharina japonica, and one EsCaM and eleven EsCMLs in Ectocarpus sp. SjCaM and EsCaM encoded identical protein products and shared 88.59–89.93% amino acid identities with Arabidopsis thaliana AtCaMs, thereby indicating that brown algae CaMs retained a similar Ca2+ sensors function as in plants. The phylogenetic and gene structure analysis results showed that there was significant divergence in the gene sequences among brown algae CMLs. Furthermore, evolutionary analysis indicated that the function of brown alga CMLs was relatively conserved, which may be related to the fact that brown algae do not need to face complex environments like terrestrial plants. Regulatory elements prediction and the expression analysis revealed the probable functioning of SjCaM/CML genes in gametophyte development and the stress response in S. japonica. In addition, the SjCaM/SjCMLs interacting proteins and chemicals were preliminarily predicted, suggesting that SjCaM/SjCMLs might play putative roles in Ca2+/CaM-mediated growth and development processes and stimulus responses. Therefore, these results will facilitate our understanding of the evolution of brown algae CaMs/CMLs and the functional identification of SjCaM/SjCMLs. [ABSTRACT FROM AUTHOR]

Background. The calmodulin-like (CML) protein is a crucial Ca2+-binding protein that can sense and conduct the Ca2+ signal in response to extracellular stimuli. TheCML protein families have been identified and characterized in many species. Nevertheless, scarce information on cucumber CML is retrievable. Methods. In this study, bioinformatic analyses, including gene structure, conserved domain, phylogenetic relationship, chromosome distribution, and gene synteny, were comprehensively performed to identify and characterize CsCML gene members. Spatiotemporal expression analysis in different organs and environment conditions were assayed with real-time quantitative polymerase chain reaction (qRT-PCR). Results. Forty-four CsCMLs family members were well characterized, and the results showed that the 44 CsCML proteins contained one to four EF-hand domains without other functional domains. Most of the CsCML proteins were intron-less and unevenly distributed on seven chromosomes; two tandemly duplicated gene pairs and three segmentally duplicated gene pairs were identified in the cucumber genome. Cis-acting element analysis showed that the hormone, stress, and plant growth and development-related elements were in the promotor regions. In addition, spatiotemporal expression analysis revealed distinctive expression patterns for CsCML genes in different tissues and environmental conditions, and a putative protein interaction network also confirmed their potential role in responding to various stimuli. These results provide a foundation for understanding CsCMLs and provide a theoretical basis for further study of the physiological functions of CsCMLs. [ABSTRACT FROM AUTHOR]

Soil salinity is a major abiotic stress that causes disastrous losses in crop yields. To identify favorable alleles that enhance the salinity resistance of rice (Oryza sativa L.) crops, a set of 120 Cheongcheong Nagdong double haploid (CNDH) lines derived from a cross between the Indica variety Cheongcheong and the Japonica variety Nagdong were used. A total of 23 QTLs for 8 different traits related to salinity resistance on chromosomes 1–3 and 5–12 were identified at the seedling stage. A QTL related to the salt injury score (SIS), qSIS-3b, had an LOD score of six within the interval RM3525–RM15904 on chromosome 3, and a phenotypic variation of 31% was further examined for the candidate genes. Among all the CNDH populations, five resistant lines (CNDH 27, CNDH 34-1, CNDH 64, CNDH 78, and CNDH 112), five susceptible lines (CNDH 52-1, CNDH 67, CNDH 69, CNDH 109, and CNDH 110), and the parent lines Cheongcheong and Nagdong were selected for relative gene expression analysis. Among all the genes, two candidate genes were highly upregulated in resistant lines, including the auxin-responsive protein IAA13 (Os03g0742900) and the calmodulin-like protein 4 (Os03g0743500-1). The calmodulin-like protein 4 (Os03g0743500-1) showed a higher expression in all the resistant lines than in the susceptible lines and a high similarity with other species in sequence alignment and phylogenetic tree, and it also showed a protein–protein interaction with other important proteins. The genes identified in our study will provide new genetic resources for improving salt resistance in rice using molecular breeding strategies in the future. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM) and calmodulin-like (CML) genes are widely involved in plant growth and development and mediating plant stress tolerance. However, the whole genome scale studies about CaM and CML gene families have not been done in wheat, and the possible functions of most wheat CaM/CML gene members are still unknown. In this study, a total of 18 TaCaM and 230 TaCML gene members were identified in wheat genome. Among these genes, 28 TaCaM/CML gene members have 74 duplicated copies, while 21 genes have 48 transcript variants, resulting in 321 putative TaCaM/CML transcripts totally. Phylogenetic tree analysis showed that they can be classified into 7 subfamilies. Similar gene structures and protein domains can be found in members of the same gene cluster. The TaCaM/CML genes were spread among all 21 chromosomes with unbalanced distributions, while most of the gene clusters contained 3 homoeologous genes located in the same homoeologous chromosome group. Synteny analysis showed that most of TaCaM/CMLs gene members can be found with 1–4 paralogous genes in T. turgidum and Ae. Tauschii. High numbers of cis-acting elements related to plant hormones and stress responses can be observed in the promoters of TaCaM/CMLs. The spatiotemporal expression patterns showed that most of the TaCaM/TaCML genes can be detected in at least one tissue. The expression levels of TaCML17, 21, 30, 50, 59 and 75 in the root or shoot can be up-regulated by abiotic stresses, suggesting that TaCML17, 21, 30, 50, 59 and 75 may be related with responses to abiotic stresses in wheat. The spatiotemporal expression patterns of TaCaM/CML genes indicated they may be involved widely in wheat growth and development. Our results provide important clues for exploring functions of TaCaMs/CMLs in growth and development as well as responses to abiotic stresses in wheat in the future.

Until now, no completely effective parasite‐specific drugs or vaccines have been approved for the treatment of cryptosporidiosis. Through the separation and identification of the sporozoite membrane protein of Cryptosporidium parvum (C. parvum), 20 related proteins were obtained. Among them, a calmodulin‐like protein (CML) has a similar functional domain‐exchange factor hand (EF‐hand) motif as calmodulin proteins (CaMs), so it may play a similarly important role in the invasion process. A 663 bp full gene encoding the C. parvum calmodulin‐like protein (CpCML) was inserted in pET28a vector and expressed in Escherichia coli. An immunofluorescence assay showed that CpCML was mainly located on the surface of the sporozoites. Three‐week‐old female BALB/c mice were used for modelling the immunoreactions and immunoprotection of recombinant CpCML (rCpCML) against artificial Cryptosporidium tyzzeri infections. The results indicated a significantly increased in anti‐CpCML antibody response, which was induced by the immunized recombinant protein. Compared to rP23 (recombinant P23), GST6P‐1 (expressed by pGEX‐6P‐1 transfected E. coli), GST4T‐1 (expressed by pGEX‐4T‐1 transfected E. coli), glutathione (GSH), adjuvant and blank control groups, rCpCML‐immunized mice produced specific spleen cell proliferation in addition to different production levels of IL‐2, IFN‐γ, TNF‐α, IL‐4 and IL‐5. Additionally, immunization with rCpCML led to 34.08% reduction of oocyst shedding in C. tyzzeri infected mice faeces which was similar to rP23. These results suggest that CpCML may be developed as a potential vaccine candidate antigen against cryptosporidiosis. [ABSTRACT FROM AUTHOR]

Calmodulins (CaMs) and Calmodulin-like proteins (CMLs) are vital in plant growth, development, and stress responses. However, CaMs and CMLs have not been fully identified and characterized in brown algae, which has been evolving independently of the well-studied green plant lineage. In this study, whole-genome searches revealed one SjCaM and eight SjCMLs in Saccharina japonica, and one EsCaM and eleven EsCMLs in Ectocarpus sp. SjCaM and EsCaM encoded identical protein products and shared 88.59–89.93% amino acid identities with Arabidopsis thaliana AtCaMs, thereby indicating that brown algae CaMs retained a similar Ca2+ sensors function as in plants. The phylogenetic and gene structure analysis results showed that there was significant divergence in the gene sequences among brown algae CMLs. Furthermore, evolutionary analysis indicated that the function of brown alga CMLs was relatively conserved, which may be related to the fact that brown algae do not need to face complex environments like terrestrial plants. Regulatory elements prediction and the expression analysis revealed the probable functioning of SjCaM/CML genes in gametophyte development and the stress response in S. japonica. In addition, the SjCaM/SjCMLs interacting proteins and chemicals were preliminarily predicted, suggesting that SjCaM/SjCMLs might play putative roles in Ca2+/CaM-mediated growth and development processes and stimulus responses. Therefore, these results will facilitate our understanding of the evolution of brown algae CaMs/CMLs and the functional identification of SjCaM/SjCMLs.

Calmodulins (CAMs) and calmodulin-like proteins (CMLs) can participate in the regulation of various physiological processes via sensing and decoding Ca2+ signals. To reveal the characteristics of the CAM/CML family in Ginkgo biloba, a comprehensive analysis was performed at the genome-wide level. A total of 26 CAMs/CMLs, consisting of 5 GbCAMs and 21 GbCMLs, was identified on 11 out of 12 chromosomes in G. biloba. They displayed a certain degree of multiplicity in their sequences, albeit with conserved EF hands. Collinearity analysis suggested that tandem rather than segmental or whole-genome duplications were likely to play roles in the evolution of the Ginkgo CAM/CML family. Furthermore, GbCAMs/GbCMLs were grouped into higher, lower, and moderate expression in magnitude. The cis-acting regulatory elements involved in phytohormone-responsiveness within GbCAM/GbCML promotors may explain their varied expression profiles. The ectopic expression of a GbCML gene (Gb_30819) in transgenic Arabidopsis led to phenotypes with significantly shortened root length and seedling height, and decreased yields of both pods and seeds. Moreover, an electrophoresis mobility shift assay demonstrated the Ca2+-binding activity of Gb_30819 in vitro. Altogether, these results contribute to insights into the characteristics of the evolution and expression of GbCAMs/GbCMLs, as well as evidence for Ca2+-CAM/CML pathways functioning within the ancient gymnosperm G. biloba. [ABSTRACT FROM AUTHOR]

Calmodulin-like proteins (CMLs) are one of the Ca2+ sensors in plants, but the functions of most CMLs remain unknown. The regulation of cold tolerance and flowering time by MtCML42 in Medicago truncatula and the underlying mechanisms were investigated using MtCML42-overexpressing plants and cml42 Medicago mutants with a Tnt1 retrotransposon insertion. Compared with the wild type (WT), MtCML42-overexpressing lines had increased cold tolerance, whereas cml42 mutants showed decreased cold tolerance. The impaired cold tolerance in cml42 could b complemented by MtCML42 expression. The transcript levels of MtCBF1, MtCBF4, MtCOR413, MtCAS15, MtLTI6A, MtGolS1 and MtGolS2 and the concentrations of raffinose and sucrose were increased in response to cold treatment, whereas higher levels were observed in MtCML42-overexpressing lines and lower levels were observed in cml42 mutants. In addition, early flowering with upregulated MtFTa1 and downregulated MtABI5 transcripts was observed in MtCML42-overexpressing lines, whereas delayed flowering with downregulated MtFTa1 and upregulated MtABI5 was observed in cml42. MtABI5 expression could complement the flowering phenotype in the Arabidopsis mutant abi5. Our results suggest that MtCML42 positively regulates MtCBF1 and MtCBF4 expression, which in turn upregulates the expression of some COR genes, MtGolS1 and MtGolS2, which leads to raffinose accumulation and increased cold tolerance. MtCML42 regulates flowering time through sequentially downregulating MtABI5 and upregulating MtFTa1 expression. [ABSTRACT FROM AUTHOR]

Pear (Pyrus bretschneideri), one of the most widely planted fruit trees in the world, is infected by pear ring rot disease, which is triggered by Botryosphaeria dothidea. Previous research has shown that exogenous calcium enhanced pear resistance to B. dothidea. To explore the molecular mechanism of calcium in pear pathogen resistance, we searched the differentially expressed genes (DEGs) between calcium and H2O treatment with B. dothidea inoculation in pear by using RNA-seq data. On the basis of the standard of a proportion of calcium/H2O fold change >2, and the false discovery rate (FDR) <0.05, 2,812 and 572 genes with significant differential expression were identified between the H2O and calcium treatments under B. dothidea inoculation at 2 days postinoculation (dpi) (D2) and 8 dpi (D8), respectively, indicating that significantly more genes in D2 responded to calcium treatment. Results of the gene annotation showed that DEGs were focused on plant-pathogen interactions, hormone signal transduction, and phenylpropanoid biosynthesis in D2. Moreover, transient silencing of PbrCML30 (pear calmodulin-like proteins 30), which had significantly higher expression in response to calcium than H2O treatments, conferred compromised resistance to B. dothidea. Exogenous calcium treatment slightly alleviated the symptoms of TRV2-PbrCML30 leaves compared with TRV2 leaves under inoculation, supporting its key role in pear resistance to B. dothidea. Overall, the information obtained in this study provides a possible mechanism of calcium in regulating pear resistance to B. dothidea. [ABSTRACT FROM AUTHOR]

Calcium signaling plays a crucial role as a second messenger in plant immune responses. However, the regulatory mechanisms that govern host responses to infections caused by Sclerotinia sclerotiorum and Phytophthora capsici remain unclear. This study investigated the expression pattern of Nicotiana benthamiana calmodulin-like protein 30 (NbCML30) following infections. Our results reveal a significant downregulation of NbCML30 expression after S. sclerotiorum infection, with no changes observed in NbCML30 expression following P. capsici infection. Importantly, transient silencing of NbCML30 using tobacco rattle virus-mediated methods promoted infections caused by both S. sclerotiorum and P. capsici. In contrast, transgenic plants overexpressing NbCML30 exhibited a suppressive effect on the infections caused by S. sclerotiorum and P. capsici. Subsequent transcriptome sequencing uncovered that NbCML30 silencing markedly upregulated the expression of host endogenous genes, particularly activating pathways associated with protein processing in endoplasmic reticulum (ER) associated degradation (ERAD) pathway. These findings highlight the affirmative regulatory role of NbCML30 in conferring resistance against necrotrophic and hemibiotrophic pathogens, shedding light on its potential inhibitory mechanism. In conclusion, our study provides a theoretical basis for crop resistance genetic breeding strategies by elucidating the regulatory function of NbCML30 in plant defense against these pathogens. • Silencing NbCML30 enhances susceptibility to S. sclerotiorum and P. capsici infections. • Silencing NbCML30 activates pathways associated with endoplasmic reticulum-associated degradation (ERAD). • NbCML30 overexpression inhibits S. sclerotiorum and P. capsici infection. [ABSTRACT FROM AUTHOR]

Soil salinity is a major abiotic stress that causes disastrous losses in crop yields. To identify favorable alleles that enhance the salinity resistance of rice (Oryza sativa L.) crops, a set of 120 Cheongcheong Nagdong double haploid (CNDH) lines derived from a cross between the Indica variety Cheongcheong and the Japonica variety Nagdong were used. A total of 23 QTLs for 8 different traits related to salinity resistance on chromosomes 1–3 and 5–12 were identified at the seedling stage. A QTL related to the salt injury score (SIS), qSIS-3b, had an LOD score of six within the interval RM3525–RM15904 on chromosome 3, and a phenotypic variation of 31% was further examined for the candidate genes. Among all the CNDH populations, five resistant lines (CNDH 27, CNDH 34-1, CNDH 64, CNDH 78, and CNDH 112), five susceptible lines (CNDH 52-1, CNDH 67, CNDH 69, CNDH 109, and CNDH 110), and the parent lines Cheongcheong and Nagdong were selected for relative gene expression analysis. Among all the genes, two candidate genes were highly upregulated in resistant lines, including the auxin-responsive protein IAA13 (Os03g0742900) and the calmodulin-like protein 4 (Os03g0743500-1). The calmodulin-like protein 4 (Os03g0743500-1) showed a higher expression in all the resistant lines than in the susceptible lines and a high similarity with other species in sequence alignment and phylogenetic tree, and it also showed a protein–protein interaction with other important proteins. The genes identified in our study will provide new genetic resources for improving salt resistance in rice using molecular breeding strategies in the future.

Copyright of Journal of Tea Science is the property of Journal of Tea Science Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Calmodulins (CAMs) and calmodulin-like proteins (CMLs) can participate in the regulation of various physiological processes via sensing and decoding Ca2+ signals. To reveal the characteristics of the CAM/CML family in Ginkgo biloba, a comprehensive analysis was performed at the genome-wide level. A total of 26 CAMs/CMLs, consisting of 5 GbCAMs and 21 GbCMLs, was identified on 11 out of 12 chromosomes in G. biloba. They displayed a certain degree of multiplicity in their sequences, albeit with conserved EF hands. Collinearity analysis suggested that tandem rather than segmental or whole-genome duplications were likely to play roles in the evolution of the Ginkgo CAM/CML family. Furthermore, GbCAMs/GbCMLs were grouped into higher, lower, and moderate expression in magnitude. The cis-acting regulatory elements involved in phytohormone-responsiveness within GbCAM/GbCML promotors may explain their varied expression profiles. The ectopic expression of a GbCML gene (Gb_30819) in transgenic Arabidopsis led to phenotypes with significantly shortened root length and seedling height, and decreased yields of both pods and seeds. Moreover, an electrophoresis mobility shift assay demonstrated the Ca2+-binding activity of Gb_30819 in vitro. Altogether, these results contribute to insights into the characteristics of the evolution and expression of GbCAMs/GbCMLs, as well as evidence for Ca2+-CAM/CML pathways functioning within the ancient gymnosperm G. biloba.

The effect of calcium chloride (CaCl 2) treatment on γ-aminobutyric acid (GABA) accumulation in fresh-cut cantaloupe and the involved mechanisms were investigated. The result showed that 1% (w/v) CaCl 2 treatment increased GABA content and activities of glutamate decarboxylase (GAD) and succinate semialdehyde dehydrogenase (SSADH), while decreased glutamate (Glu) content and GABA transaminase (GABA-T) activities in fresh-cut cantaloupe. CmCML11 and CmCAMTA5 expressions of CaCl 2 -treated fruit increased by 187.4% and 165.6% than control fruit in the initial 6 h. Besides, expressions of GABA shunt genes, including CmGAD1 , CmGAD2 , CmGABA-T and CmSSADH were also up-regulated by CaCl 2 treatment during early storage. Moreover, acting as a transcriptional activator, CmCAMTA5 could bind to the CG-box in promoters of CmGAD1 , CmGABA-T and CmSSADH and activate their transcription. Furthermore, the interaction between CmCML11 and CmCAMTA5 could enhance the transcriptional activation on GABA shunt genes which were regulated by CmCAMTA5. Collectively, our findings revealed that CaCl 2 treatment promoted GABA accumulation in fresh-cut cantaloupe via the combined effect of CmCML11 and CmCAMTA5 in the regulation of expressions of CmGAD1 , CmGABA-T , and CmSSADH in GABA shunt. [Display omitted] • CaCl 2 treatment could activate GABA shunt and enhance GABA content of fresh-cut cantaloupe. • CaCl 2 treatment up-regulated the expressions of CmCML11 and CmCAMTA5. • CmCML11 protein physically interacted with CmCAMTA5. • GABA shunt was positively regulated by Ca2+-CML11-CAMTA5 signal transduction pathway. [ABSTRACT FROM AUTHOR]

[Objectives] Calmodulin-like protein (CML), a class of Ca2+ binding proteins containing the EF-hand domain, are closely related to the division and morphogenesis of plant cells. Based on the cloned CML25-like gene which expressed specifically in the cucumber fruit, we studied its mechanism of regulating parthenocarpy. [Methods] RT-qPCR method was used to study the temporal and spatial characteristics of CML25-like expression. Plant expression vector pLP100-35S-CML25-like was constructed, the cucumber inbred line CCMC was transformed by Agrobacterium-mediated transformation method using cotyledon as explants, and the expression characteristics of the transgenic plants were studied. The phenotypes of transgenic plants were observed, and the expression changes of hormone-related genes in fruit-set period of transgenic plants were detected using RT-qPCR method. [Results] In transgenic plants, the expression level of CML25-like was the highest in young leaves and female flowers of cucumber, and the lowest in male flowers. Moreover, CML25-like was up-regulated expression during the fruit development, and down-regulated expression during the fruit abortion. Statistical results of traits showed that the parthenocarpy rate of transgenic plants of over-expressing CML25-like could reach to 98.25%, which increased 86% compared with control CCMC. And there were no significant differences in length and transverse diameter between the parthenocarpic fruits and control pollinated fruits. The results of RT-qPCR showed that 16 of the 22 hormone-related genes were significant up-regulated expression during the fruit setting of transgenic plants. [Conclusions] The active expression of CML25-like was closely related to fruit development, and CML25-like could induce parthenocarpy of cucumber and promote fruit enlargement. We inferred that CML25-like induced parthenocarpy by promoting the up-regulated expression of hormone-related genes in the ovaries of weakly parthenocarpic cucumber. [ABSTRACT FROM AUTHOR]

Main conclusion: The molecular mechanisms regulating calcium-mediated thermotolerance in Camellia sinensis were revealed by RNA-Sequencing.Heat stress is one of the most remarkable abiotic factors limiting the growth and productivity of Camellia sinensis plants. Calcium helps regulate plant responses to various adverse environmental conditions, including heat stress. In this study, the effects of exogenous calcium on the physiological characteristics of heat-stressed C. sinensis were investigated. A calcium pretreatment increased the proline, soluble sugar, Ca2+, and chlorophyll contents, but decreased the malondialdehyde content and relative electrical conductivity in C. sinensis leaves under heat stress. Further analysis of the ultra-structure of chloroplasts indicated that heat stress induced accumulation of starch granules and destruction of the stroma lamella in C. sinensis. However, calcium pretreatment counteracted the adverse effects of heat stress on the structure of the photosynthetic apparatus. These results imply that the calcium pretreatment increased C. sinensis thermotolerance. Moreover, RNA-sequencing was applied to characterize the calcium-mediated transcript-level responses to heat stress. A total of 923 differentially expressed genes (DEGs) including 299 up-regulated and 624 down-regulated genes were identified. Functional annotations indicated that these DEGs were primarily related to signal transduction, transcriptional regulation, and post-translational modification. In addition, a C. sinensis gene [CsCML45 (GenBank: KY652927)] encoding a calmodulin-like protein was isolated. The heterologous expression of CsCML45 enhanced the thermotolerance of transgenic Arabidopsis thaliana plants. These results may be useful for characterizing the calcium-mediated molecular mechanism responsible for C. sinensis thermotolerance. [ABSTRACT FROM AUTHOR]

The calmodulin (CaM) and calmodulin-like (CML) proteins are major calcium sensors that play a critical role in environmental stimulus response in plants. Nevertheless, the CaM/CML proteins from the specific plants with extreme tolerance to abiotic stresses remained so far uncharacterized. In this study, 66 candidate proteins (three NsCaMs and sixty-three NsCMLs) were identified from the halophyte Nitraria sibirica , which can withstand an extreme salinity. Bioinformatic analysis of upstream cis -acting elements predicted the potential involvement of NsCaM/CMLs in abiotic stress responses and various hormone responses. Additionally, the Nitraria sibirica transcriptome revealed that 17 and 7 NsCML s were significantly upregulated under 100 mM or 400 mM NaCl treatment. Transcription of most salt-responsive genes was similarly upregulated under cold stress, yet downregulated under drought treatment. Moreover, predictive subcellular localization analysis suggested that the stress-responsive NsCML proteins mainly localize at the cellular membrane and within the nucleus. Furthermore, transgenic overexpression of two NsCML s (NISI03G1136 and NISI01G1645) was found to mitigate H 2 O 2 accumulation caused by salt stress. These results provide insights into the potential function of Nitraria sibirica CaM/CML proteins, which could aid the investigation of molecular mechanisms of extreme tolerance to abiotic stresses in halophytes. [ABSTRACT FROM AUTHOR]

Abstract In plants, many target proteins of calmodulins (CaMs) have been identified in cellular metabolism and responses. However, calmodulin-like proteins (CMLs) and their target proteins have not been discovered in stress responses in rice. In this study, a novel CC-NBS-LRR protein was obtained in screening a cold stress rice seedlings yeast cDNA library with OsCML16 as bait. Furthermore, yeast two-hybrid and BiFC assays demonstrated that the full length, CC region in the N-terminus and LRR in the C-terminus of Pi304 protein could interact with OsCML16. More interestingly, OsCML16 bound to the 1–10 motif rather than 1–14 motif in the Ca2+ or Mg2+ dependent manner in vitro. In addition, transcript levels of OsCML16 and OsPi304 were induced more markedly in Nipponbare than in 9311 under cold stress. Taken together, these data indicates that they are involved in the cold stress signaling and response in rice. Highlights • OsCML16 binds to both Ca2+ and Mg2+ in vitro. • OsCML16 interacts with a novel CC-NBS-LRR protein OsPi304. • OsCML16 binds to the 1-10 motif in a Ca2+/Mg2+-dependent manner in vitro. • OsCML16 binds to LRR domain in a Ca2+/Mg2+-independent manner in vitro. • OsPi304 and OsCML16 genes are induced by cold stress. [ABSTRACT FROM AUTHOR]

Calmodulin-like protein 19 (CML19) is an Arabidopsis centrin that modulates nucleotide excision repair (NER) by binding to RAD4 protein, the Arabidopsis homolog of human Xeroderma pigmentosum complementation group C protein. Although the necessity of CML19 as a part of the RAD4 plant recognition complex for functional NER is known at a cellular level, little is known at a molecular level. Herein, we used a combination of biophysical and biochemical approaches to investigate the structural and ion and target-peptide binding properties of CML19. We found that CML19 possesses four Ca 2+ -specific binding sites, two of high affinity in the N-terminal domain and two of low affinity in the C-terminal domain. Binding of Ca 2+ to CML19 increases its alpha-helix content, stabilizes the tertiary structure, and triggers a conformational change, resulting in the exposure of a hydrophobic patch instrumental for target protein recognition. Using bioinformatics tools we identified a CML19-binding site at the C-terminus of RAD4, and through in vitro binding experiments we analyzed the interaction between a 17-mer peptide representing this site and CML19. We found that the peptide shows a high affinity for CML19 in the presence of Ca 2+ (stoichiometry 1:1) and the interaction primarily involves the C-terminal half of CML19. [ABSTRACT FROM AUTHOR]

Calcium signalling, including pulse, amplitude, and duration, is essential for plant development and response to various stimuli. However, the calcium signalling should be decoded and translated by calcium sensors. In plants, three classes of calcium-binding proteins have been identified as calcium sensors, including calcium-dependent protein kinase (CDPK), calcineurin B-like protein (CBL), and calmodulin (CaM). Calmodulin-like proteins (CMLs), which have several EF-hands, also serve as specific calcium sensors and can sense, bind, and interpret the calcium signal during the plant's growth and defense decision-making processes. In recent decades, the function of CMLs in plant development and response to various stimuli has been systematically reviewed, shedding light on the molecular mechanism of plant CML-mediated networks in calcium signal transduction. Here, by providing an overview of CML expression and biological function in plants, we demonstrate that growth-defense trade-offs occur during calcium sensing, an aspect that has not been well studied in recent years. [ABSTRACT FROM AUTHOR]

The AP2/ ERF family is a plant-specific transcription factor family whose members have been associated with various developmental processes and stress tolerance. Here, we functionally characterized the drought-inducible Os ERF48, a group Ib member of the rice ERF family with four conserved motifs, CMI-1, -2, -3 and -4. A transactivation assay in yeast revealed that the C-terminal CMI-1 motif was essential for Os ERF48 transcriptional activity. When Os ERF48 was overexpressed in an either a root-specific ( ROX O s ERF 48) or whole-body ( OX O s ERF 48) manner, transgenic plants showed a longer and denser root phenotype compared to the nontransgenic ( NT) controls. When plants were grown on a 40% polyethylene glycol-infused medium under in vitro drought conditions, ROX O s ERF 48 plants showed a more vigorous root growth than OX O s ERF 48 and NT plants. In addition, the ROX O s ERF 48 plants exhibited higher grain yield than OX O s ERF 48 and NT plants under field-drought conditions. We constructed a putative Os ERF48 regulatory network by cross-referencing ROX O s ERF 48 root-specific RNA-seq data with a co-expression network database, from which we inferred the involvement of 20 drought-related genes in Os ERF48-mediated responses. These included genes annotated as being involved in stress signalling, carbohydrate metabolism, cell-wall proteins and drought responses. They included, Os CML16, a key gene in calcium signalling during abiotic stress, which was shown to be a direct target of Os ERF48 by chromatin immunoprecipitation- qPCR analysis and a transient protoplast expression assay. Our results demonstrated that Os ERF48 regulates Os CML16, a calmodulin-like protein gene that enhances root growth and drought tolerance. [ABSTRACT FROM AUTHOR]

Primary infection of a plant with a pathogen that causes high accumulation of salicylic acid in the plant typically via a hypersensitive response confers enhanced resistance against secondary infection with a broad spectrum of pathogens, including viruses. This phenomenon is called systemic acquired resistance (SAR), which is a plant priming for adaption to repeated biotic stress. However, the molecular mechanisms of SAR-mediated enhanced inhibition, especially of virus infection, remain unclear. Here, we show that SAR against cucumber mosaic virus (CMV) in tobacco plants (Nicotiana tabacum) involves a calmodulin-like protein, rgs-CaM. We previously reported the antiviral function of rgs-CaM, which binds to and directs degradation of viral RNA silencing suppressors (RSSs), including CMV 2b, via autophagy. We found that rgs-CaM-mediated immunity is ineffective against CMV infection in normally growing tobacco plants but is activated as a result of SAR induction via salicylic acid signaling. We then analyzed the effect of overexpression of rgs- CaM on salicylic acid signaling. Overexpressed and ectopically expressed rgs-CaM induced defense reactions, including cell death, generation of reactive oxygen species, and salicylic acid signaling. Further analysis using a combination of the salicylic acid analogue benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and the Ca2+ ionophore A23187 revealed that rgs-CaM functions as an immune receptor that induces salicylic acid signaling by simultaneously perceiving both viral RSS and Ca2+ influx as infection cues, implying its autoactivation. Thus, secondary infection of SAR-induced tobacco plants with CMV seems to be effectively inhibited through 2b recognition and degradation by rgs-CaM, leading to reinforcement of antiviral RNA silencing and other salicylic acid-mediated antiviral responses. IMPORTANCE Even without an acquired immune system like that in vertebrates, plants show enhanced whole-plant resistance against secondary infection with pathogens; this so-called systemic acquired resistance (SAR) has been known for more than half a century and continues to be extensively studied. SAR-induced plants strongly and rapidly express a number of antibiotics and pathogenesis-related proteins targeted against secondary infection, which can account for enhanced resistance against bacterial and fungal pathogens but are not thought to control viral infection. This study showed that enhanced resistance against cucumber mosaic virus is caused by a tobacco calmodulin-like protein, rgs-CaM, which detects and counteracts the major viral virulence factor (RNA silencing suppressor) after SAR induction. rgs-CaM-mediated SAR illustrates the growth versus defense trade-off in plants, as it targets the major virulence factor only under specific biotic stress conditions, thus avoiding the cost of constitutive activation while reducing the damage from virus infection. [ABSTRACT FROM AUTHOR]

L. japonicus, a model plant of legumes plants, is widely used in symbiotic nitrogen fixation. A large number of studies on it have been published based on the genetic, biochemical, structural studies. These results are secondhand reports that CaM is a key regulator during Rhizobial infection. In plants, there are multiple CaM genes encoding several CaM isoforms with only minor amino acid differences. Moreover, the regulation mechanism of this family of proteins during rhizobia infection is still unclear. In the current study, a family of genes encoding CaMs and CMLs that possess only the Ca2+-binding EF-hand motifs were analyzed. Using ML and BI tree based on amino acid sequence similarity, seven loci defined as CaMs and 19 CMLs, with at least 23% identity to CaM, were identified. The phylogenetics, gene structures, EF hand motif organization, and expression characteristics were evaluated. Seven CaM genes, encoding only 4 isoforms, were found in L. japonicus. According to qRT-PCR, four LjCaM isoforms are involved in different rhizobia infection stages. LjCaM1 might be involved in the early rhizobia infection epidermal cells stage. Furthermore, additional structural differences and expression behaviors indicated that LjCMLs may have different potential functions from LjCaMs. [ABSTRACT FROM AUTHOR]

• A novel CgCaLP-2 gene was identified in Crassostrea gigas and it underwent alternative splicing. • Two protein isoforms were detected and the 23 kD form mainly responded to long-term CO2 exposure. • CgCaLP-2 redistributed to nuclear membrane after long-term CO2 exposure. • The expression of a transcription factor CgSIX4 was associated with CgCaLP-2 under CO2 exposure and in vivo knock down further verified their relevance. Marine organisms need to adapt to improve organismal fitness under ocean acidification (OA). Recent studies have shown that marine calcifiers can achieve acclimation by stimulating calcium binding/signaling pathways. Here, a CaM-like gene (CgCaLP-2) from oyster Crassostrea gigas which typically responded to long-term CO 2 exposure (two months) rather than short-term exposure (one week) was characterized. The cloned cDNA was 678 bp and was shorter than the retrieved sequence from NCBI (1125 bp). The two sequences, designated as CgCaLP-2-v1 and CgCaLP-2-v2 , were demonstrated to be different splice variants by the genome sequence analysis. Western blotting analysis revealed two bands of 23 kD and 43 kD in mantle and hemocytes, corresponding to predicted molecular weight of CgCaLP-2-v1 and CgCaLP-2-v2, respectively. The isoform CgCaLP-2-v1 (the 23 kD band) was highly stimulated in response to long-term CO 2 exposure (42-day and 56-day treatment) in hemocytes and mantle tissue. The fluorescence signal of CgCaLP-2 in mantle and hemocytes became more intensive after long-term CO 2 exposure. Besides, in hemocytes, CgCaLP-2 presented a higher localization on the nuclear membrane after long-term CO 2 exposure (56 d). The target gene network of CgCaLP-2 was predicted, and a transcription factor (TF) gene annotated as Homeobox protein SIX4 (CgSIX4) showed a similar expressive trend to CgCaLP-2 during CO 2 exposure. Suppression of CgCaLP-2 via RNA interference significantly reduced the mRNA expression of CgSIX4. The results suggested that CgCaLP-2 might mediate the Ca2+-CaLP-TF signal transduction pathway under long-term CO 2 exposure. This study serves as an example to reveal that alternative splicing is an important mechanism for generation multiple protein isoforms and thus shape the plastic responses under CO 2 exposure, providing new insight into the potential acclimation ability of marine calcifiers to future OA. [ABSTRACT FROM AUTHOR]

Transient changes in intracellular Ca2+ concentration have been well recognized to act as cell signals coupling various environmental stimuli to appropriate physiological responses with accuracy and specificity in plants. Calmodulin (CaM) and calmodulin-like proteins (CMLs) are major Ca2+ sensors, playing critical roles in interpreting encrypted Ca2+ signals. Ca2+-loaded CaM/CMLs interact and regulate a broad spectrum of target proteins such as channels/pumps/antiporters for various ions, transcription factors, protein kinases, protein phosphatases, metabolic enzymes and proteins with unknown biochemical functions. Many of the target proteins of CaM/CMLs directly or indirectly regulate plant responses to environmental stresses. Basic information about stimulus-induced Ca2+ signal and overview of Ca2+ signal perception and transduction are briefly discussed in the beginning of this review. How CaM/CMLs are involved in regulating plant responses to abiotic stresses are emphasized in this review. Exciting progress has been made in the past several years, such as the elucidation of Ca2+/CaM-mediated regulation of AtSR1/CAMTA3 and plant responses to chilling and freezing stresses, Ca2+/CaM-mediated regulation of CAT3, MAPK8 and MKP1 in homeostasis control of ROS signals, discovery of CaM7 as a DNA-binding transcription factor regulating plant response to light signals. However, many key questions in Ca2+/CaM-mediated signaling warrant further investigation. Ca2+/CaM-mediated regulation of most of the known target proteins is presumed based on their interaction. The downstream targets of CMLs are mostly unknown, and how specificity of Ca2+ signaling could be realized through the actions of CaM/CMLs and their target proteins is largely unknown. Future breakthroughs in Ca2+/CaM-mediated signaling will not only improve our understanding of how plants respond to environmental stresses, but also provide the knowledge base to improve stress-tolerance of crops.

The tobacco calmodulin-like protein rgs-CaM is involved in host defense against virus and is reported to possess an associated RNA silencing suppressor activity. Rgs-CaM is also believed to act as an antiviral factor by interacting and targeting viral silencing suppressors for autophagic degradation. Despite these functional data, calcium interplay in the modulation of rgs-CaM is still poorly understood. Here we show that rgs-CaM displays a prevalent alpha-helical conformation and possesses three functional Ca 2+ -binding sites. Using computational modeling and molecular dynamics simulation, we demonstrate that Ca 2+ binding to rgs-CaM triggers expansion of its tertiary structure with reorientation of alpha-helices within the EF-hands. This conformational change leads to the exposure of a large negatively charged region that may be implicated in the electrostatic interactions between rgs-CaM and viral suppressors. Moreover, the k d values obtained for Ca 2+ binding to the three functional sites are not within the affinity range of a typical Ca 2+ sensor. [ABSTRACT FROM AUTHOR]

In addition to the well-known Ca2+ sensor calmodulin, plants possess many calmodulin-like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca2+ and Mg2+ binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS-based fluorescence, to evaluate the structural effects of metal binding and metal-induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca2+ ion with micromolar affinity ( Kd ∼ 12 µ M) and the presence of 10 m M Mg2+ decreases the Ca2+ affinity by ∼5-fold. Although binding of Ca2+ to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca2+ sensors, but causes only localized structural changes in the unique functional EF-hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role. [ABSTRACT FROM AUTHOR]

Both calcium ion (Ca 2+ ) and salicylic acid (SA) influence various stress responses in plants. In acidic soils, aluminum (Al) toxicity adversely affects crop yield. In this study, we determined the influences of Ca 2+ and SA on root elongation, Al accumulation, and citrate secretion in soybean plant. We also investigated the activity of antioxidative enzymes in Al-exposed soybean roots. Root elongation was severally inhibited when the roots were exposed to 30 μM Al. The Al-induced inhibition of root elongation was ameliorated by Ca 2+ and SA but aggravated by Ca 2+ channel inhibitor (VP), CaM antagonists (TFP), Ca 2+ chelator (EGTA), and SA biosynthesis inhibitor (PAC). Furthermore, 1.0 mM CaCl 2 and 10 μM SA reduced the accumulation of Al in roots, but their inhibitors stimulated the accumulation of Al in roots. Citrate secretion from these roots increased with the addition of either 1.0 mM CaCl 2 or 10 μM SA but did not increase significantly when treated with higher Ca 2+ concentration. Enzymatic analysis showed that Ca 2+ and SA stimulated the activities of superoxidase (SOD), peroxidase (POD), and ascorbate peroxidase (APX) in Al-treated roots. In addition, SA restored the inhibition of Ca 2+ inhibitors on root elongation and Al content. Thus, both Ca 2+ and SA contribute to Al tolerance in soybean. Furthermore, Ca 2+ supplements rapidly increased Al-induced accumulation of free-SA or conjugated SA (SAG), while Ca 2+ inhibitors delayed the accumulation of SA for more than 8 h. Within 4 h of treatment, SA increased cytosolic Ca 2+ concentration in Al-treated roots, and upregulated the expression of four genes that possibly encode calmodulin-like (CML) proteins. These findings indicate that SA is involved in Ca 2+ -mediated signal transduction pathways in Al tolerance. [ABSTRACT FROM AUTHOR]

Background: The calmodulin-like (CML) protein is a crucial Ca 2+ -binding protein that can sense and conduct the Ca 2+ signal in response to extracellular stimuli. The CML protein families have been identified and characterized in many species. Nevertheless, scarce information on cucumber CML is retrievable., Methods: In this study, bioinformatic analyses, including gene structure, conserved domain, phylogenetic relationship, chromosome distribution, and gene synteny, were comprehensively performed to identify and characterize CsCML gene members. Spatiotemporal expression analysis in different organs and environment conditions were assayed with real-time quantitative polymerase chain reaction (qRT-PCR)., Results: Forty-four CsCMLs family members were well characterized, and the results showed that the 44 CsCML proteins contained one to four EF-hand domains without other functional domains. Most of the CsCML proteins were intron-less and unevenly distributed on seven chromosomes; two tandemly duplicated gene pairs and three segmentally duplicated gene pairs were identified in the cucumber genome. Cis -acting element analysis showed that the hormone, stress, and plant growth and development-related elements were in the promotor regions. In addition, spatiotemporal expression analysis revealed distinctive expression patterns for CsCML genes in different tissues and environmental conditions, and a putative protein interaction network also confirmed their potential role in responding to various stimuli. These results provide a foundation for understanding CsCML s and provide a theoretical basis for further study of the physiological functions of CsCML s., Competing Interests: The authors declare there are no competing interests., (©2023 Liu et al.)

The concentration alteration of cytosolic-free calcium ([ Ca2+]cyt) is a well-known secondary messenger in plants and plays important roles during pollen grain germination and tube elongation. Here we demonstrate that CML25, a member of calmodulin-like proteins, has Ca2+-binding activity and plays a role in pollen grain germination, tube elongation and seed setting. CML 25 transcript was abundant in mature pollen grains and pollen tubes, and its product CML25 protein was primarily directed to the cytoplasm. Two independent CML 25 loss-of-function T-DNA insertion mutants suffered a major reduction in both the rate of pollen germination and the elongation of the pollen tube. Also, pollen grains of cml25 mutants were less sensitive to the external K+ and Ca2+ concentration than wild-type pollen. The disruption of CML 25 increased the [ Ca2+]cyt in both the pollen grain and the pollen tube, which in turn impaired the Ca2+-dependent inhibition of whole-cell inward K+ currents in protoplasts prepared from these materials (pollen grain and pollen tube). Complementation of cml25-1 mutant resulted in the recovery of wild-type phenotype. Our findings indicate that CML25 is an important transducer in the Ca2+-mediated regulation of K+ influx during pollen germination and tube elongation. [ABSTRACT FROM AUTHOR]

Transient changes in intracellular Ca2+ concentration have been well recognized to act as cell signals coupling various environmental stimuli to appropriate physiological responses with accuracy and specificity in plants. Calmodulin (CaM) and calmodulin-like proteins (CMLs) are major Ca2+ sensors, playing critical roles in interpreting encrypted Ca2+ signals. Ca2+-loaded CaM/CMLs interact and regulate a broad spectrum of target proteins such as channels/pumps/antiporters for various ions, transcription factors, protein kinases, protein phosphatases, metabolic enzymes, and proteins with unknown biochemical functions. Many of the target proteins of CaM/CMLs directly or indirectly regulate plant responses to environmental stresses. Basic information about stimulus-induced Ca2+ signal and overview of Ca2+ signal perception and transduction are briefly discussed in the beginning of this review. How CaM/CMLs are involved in regulating plant responses to abiotic stresses are emphasized in this review. Exciting progress has been made in the past several years, such as the elucidation of Ca2+/CaM-mediated regulation of AtSR1/CAMTA3 and plant responses to chilling and freezing stresses, Ca2+/CaM-mediated regulation of CAT3, MAPK8 and MKP1 in homeostasis control of reactive oxygen species signals, discovery of CaM7 as a DNA-binding transcription factor regulating plant response to light signals. However, many key questions in Ca2+/CaM-mediated signaling warrant further investigation. Ca2+/CaM-mediated regulation of most of the known target proteins is presumed based on their interaction. The downstream targets of CMLs are mostly unknown, and how specificity of Ca2+ signaling could be realized through the actions of CaM/CMLs and their target proteins is largely unknown. Future breakthroughs in Ca2+/CaM-mediated signaling will not only improve our understanding of how plants respond to environmental stresses, but also provide the knowledge base to improve stress-tolerance of crops. [ABSTRACT FROM AUTHOR]

Cell proliferation requires calmodulin, a protein that regulates calcium-dependent enzymes involved in signal transduction pathways in eukaryotic cells. Calmodulin-like protein (CLIP) is found in certain epithelial cell types, including normal breast epithelium, and, although it closely resembles calmodulin in amino acid sequence, CLIP interacts with different proteins than does calmodulin. The observation that CLIP mRNA expression is dramatically reduced in transformed breast epithelial cells led to two hypotheses: (1) CLIP helps to maintain the differentiated state in epithelial cells; and (2) downregulation of CLIP accompanies malignant transformation of breast epithelial cells. The objective of this study was to determine if the expression of CLIP in human breast cancer specimens is reduced in comparison to its expression in normal breast tissue. Eighty human breast cancer biopsy specimens were analyzed immunohistochemically for CLIP expression by using a polyclonal rabbit antihuman CLIP antibody. CLIP expression was reduced in 79% to 88% of the invasive ductal carcinoma and lobular carcinoma specimens and in a similar fraction of the ductal carcinoma in-situ specimens, compared with normal breast specimens. None of the breast cancer specimens showed an increase in CLIP expression. These findings support the hypotheses that CLIP behaves as a functional tumor suppressor protein and is downregulated early in breast cancer progression.

Cryptosporidium parvum is an obligate protozoan parasite invading epithelial cells of small intestine of human and animals, and causing diarrheal disease. In apicomplexan parasites, calcium signaling can regulate many essential biological processes such as invasion and migration. As the main intracellular receptor for calcium ions, calmodulins control the activities of hundreds of enzymes and proteins. Calmodulin-like protein (CML) is an important member of the calmodulin family and may play a key role in C. parvum , however, the actual situation is still not clear. The present study aimed to identify the parasite interaction partner proteins of C. parvum calmodulin-like protein (Cp CML). By constructing the cpcml bait plasmid, 5 potential Cp CML - interacting proteins in C. parvum oocyst were screened by yeast-two-hybrid system (Y2H). Bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) were performed as subsequent validations. Fibrillarin RNA methylase (FBL) was identified via this screening method as Cp CML interacting protein in C. parvum. The identification of this interaction made it possible to get a further understanding of the function of Cp CML and its contribution to the pathogenicity of C. parvum. [Display omitted] • Screening of the potentially interacting proteins of Cp CML in C. parvum. • Fibrillarin RNA methylase (FBL) was identified as one of the interacting proteins of Cp CML in C. parvum. • Fibrillarin RNA methylase (FBL) may mediate 2′-O-methylation of rRNA and assistant in the maturation of rRNA in C. parvum. [ABSTRACT FROM AUTHOR]