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Summary 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 OsERF48, 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 OsERF48 transcriptional activity. When OsERF48 was overexpressed in an either a root‐specific (ROXOsERF48) or whole‐body (OXOsERF48) 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, ROXOsERF48 plants showed a more vigorous root growth than OXOsERF48 and NT plants. In addition, the ROXOsERF48 plants exhibited higher grain yield than OXOsERF48 and NT plants under field‐drought conditions. We constructed a putative OsERF48 regulatory network by cross‐referencing ROXOsERF48 root‐specific RNA‐seq data with a co‐expression network database, from which we inferred the involvement of 20 drought‐related genes in OsERF48‐mediated responses. These included genes annotated as being involved in stress signalling, carbohydrate metabolism, cell‐wall proteins and drought responses. They included, OsCML16, a key gene in calcium signalling during abiotic stress, which was shown to be a direct target of OsERF48 by chromatin immunoprecipitation‐qPCR analysis and a transient protoplast expression assay. Our results demonstrated that OsERF48 regulates OsCML16, a calmodulin‐like protein gene that enhances root growth and drought tolerance.

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.

Calmodulins (CaMs) regulate numerous Ca2+-mediated cellular processes in plants by interacting with their respective downstream effectors. Due to the limited number of CaMs, other calcium sensors modulate the regulation of Ca2+-mediated cellular processes that are not managed by CaMs. Of 50 CaM-like (CML) proteins identified in Arabidopsis thaliana, we characterized the function of CML10. Yeast two-hybrid screening revealed phosphomannomutase (PMM) as a putative interaction partner of CML10. In vitro and in vivo interaction assays were performed to analyze the interaction mechanisms of CML10 and PMM. PMM activity and the phenotypes of cml10 knock-down mutants were studied to elucidate the role(s) of the CML10–PMM interaction. PMM interacted specifically with CML10 in the presence of Ca2+ through its multiple interaction motifs. This interaction promoted the activity of PMM. The phenotypes of cml10 knock-down mutants were more sensitive to stress conditions than wild-type plants, corresponding with the fact that PMM is an enzyme which modulates the biosynthesis of ascorbic acid, an antioxidant. The results of this research demonstrate that a calcium sensor, CML10, which is an evolutionary variant of CaM, modulates the stress responses in Arabidopsis by regulating ascorbic acid production.

Transient changes in intracellular Ca(2+) 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 Ca(2+) sensors, playing critical roles in interpreting encrypted Ca(2+) signals. Ca(2+)-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 Ca(2+) signal and overview of Ca(2+) 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 Ca(2+)/CaM-mediated regulation of AtSR1/CAMTA3 and plant responses to chilling and freezing stresses, Ca(2+)/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 Ca(2+)/CaM-mediated signaling warrant further investigation. Ca(2+)/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 Ca(2+) signaling could be realized through the actions of CaM/CMLs and their target proteins is largely unknown. Future breakthroughs in Ca(2+)/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.

Ca2+ ions play a key role in a wide variety of environmental responses and developmental processes in plants, and several protein families with Ca2+-binding domains have evolved to meet these needs, including calmodulin (CaM) and calmodulin-like proteins (CMLs). These proteins have no catalytic activity, but rather act as sensor relays that regulate downstream targets. While CaM is well-studied, CMLs remain poorly characterized at both the structural and functional levels, even if they are the largest class of Ca2+ sensors in plants. The major structural theme in CMLs consists of EF-hands, and variations in these domains are predicted to significantly contribute to the functional versatility of CMLs. Herein, we focus on recent advances in understanding the features of CMLs from biochemical and structural points of view. The analysis of the metal binding and structural properties of CMLs can provide valuable insight into how such a vast array of CML proteins can coexist, with no apparent functional redundancy, and how these proteins contribute to cellular signaling while maintaining properties that are distinct from CaM and other Ca2+ sensors. An overview of the principal techniques used to study the biochemical properties of these interesting Ca2+ sensors is also presented.

The calcium (Ca 2+ ) signaling is a crucial event during plant-herbivore interaction, which involves a transient change in cytosolic Ca 2+ concentration, which is sensed by Ca 2+ -sensors, and the received message is transduced to downstream target proteins leading to appropriate defense response. Calmodulin-like proteins ( CMLs ) are calcium-sensing plant-specific proteins. Although CMLs have been identified in a few plants, they remained uncharacterized in leguminous crop plants. Therefore, a wide-range analysis of CMLs of soybean was performed, which identified 41 true CMLs with greater than 50% similarity with Arabidopsis CMLs . The phylogenetic study revealed their evolutionary relatedness with known CMLs . Further, the identification of conserved motifs, gene structure analysis, and identification of cis -acting elements strongly supported their identity as members of this family and their involvement in stress responses. Only a few Glycine max CMLs (GmCMLs) exhibited differential expression in different tissue types, and rest of them had minimal expression. Additionally, differential expression patterns of GmCMLs were observed during Spodoptera litura -feeding, wounding, and signaling compound treatments, indicating their role in plant defense. The three-dimensional structure prediction, identification of interactive domains, and docking with Ca 2+ ions of S. litura -inducible GmCMLs, indicated their identity as calcium sensors. This study on the characterization of GmCMLs provided insights into their roles in calcium signaling and plant defense during herbivory.

Background Calmodulins ( CaMs )/CaM-like proteins (CMLs) are crucial Ca 2+ -binding sensors that can decode and transduce Ca 2+ signals during plant development and in response to various stimuli. The CaM/CML gene family has been characterized in many plant species, but this family has not yet been characterized and analyzed in peanut, especially for its functions in response to Ralstonia solanacearum . In this study, we performed a genome-wide analysis to analyze the CaM/CML genes and their functions in resistance to R. solanacearum . Results Here, 67, 72, and 214 CaM/CML genes were identified from Arachis duranensis , Arachis ipaensis , and Arachis hypogaea , respectively. The genes were divided into nine subgroups (Groups I-IX) with relatively conserved exon‒intron structures and motif compositions. Gene duplication, which included whole-genome duplication, tandem repeats, scattered repeats, and unconnected repeats, produced approximately 81 pairs of homologous genes in the AhCaM/CML gene family. Allopolyploidization was the main reason for the greater number of AhCaM/CML members. The nonsynonymous (Ka) versus synonymous (Ks) substitution rates (less than 1.0) suggested that all homologous pairs underwent intensive purifying selection pressure during evolution. AhCML69 was constitutively expressed in different tissues of peanut plants and was involved in the response to R. solanacearum infection. The AhCML69 protein was localized in the cytoplasm and nucleus. Transient overexpression of AhCML69 in tobacco leaves increased resistance to R. solanacearum infection and induced the expression of defense-related genes, suggesting that AhCML69 is a positive regulator of disease resistance. Conclusions This study provides the first comprehensive analysis of the AhCaM/CML gene family and potential genetic resources for the molecular design and breeding of peanut bacterial wilt resistance.

The plant-specific mildew resistance locus O (MLO) proteins, which contain seven transmembrane domains and a conserved calmodulin-binding domain, play important roles in many plant developmental processes. However, their mechanisms that regulate plant development remain unclear. Here, we report the functional characterization of the MLO4 protein in Arabidopsis roots. The MLO4 was identified as interacting with CML12 in a screening for the interaction between the proteins from Arabidopsis MLO and calmodulin/calmodulin-like (CaM/CML) families using yeast two hybrid (Y2H) assays. Then, the interaction between MLO4 and CML12 was further verified by Luciferase Complementation Imaging (LCI) and Bimolecular Fluorescence Complementation (BiFC) assays. Genetic analysis showed that the mlo4, cml12, and mlo4 cml12 mutants displayed similar defects in root gravity response. These results imply that the MLO4 might play an important role in root gravity response through interaction with CML12. Moreover, our results also demonstrated that the interaction between the MLO and CaM/CML families might be conservative.

Background Ca 2+ ion is a versatile second messenger that operate in a wide ranges of cellular processes that impact nearly every aspect of life. Ca 2+ regulates gene expression and biotic and abiotic stress responses in organisms ranging from unicellular algae to multi-cellular higher plants through the cascades of calcium signaling processes. Results In this study, we deciphered the genomics and evolutionary aspects of calcium signaling event of calmodulin ( CaM ) and calmodulin like- ( CML ) proteins. We studied the CaM and CML gene family of 41 different species across the plant lineages. Genomic analysis showed that plant encodes more calmodulin like-protein than calmodulins. Further analyses showed, the majority of CMLs were intronless, while CaMs were intron rich. Multiple sequence alignment showed, the EF-hand domain of CaM contains four conserved D-x-D motifs, one in each EF-hand while CMLs contain only one D-x-D-x-D motif in the fourth EF-hand. Phylogenetic analysis revealed that, the CMLs were evolved earlier than CaM and later diversified. Gene expression analysis demonstrated that different CaM and CMLs genes were express differentially in different tissues in a spatio-temporal manner. Conclusion In this study we provided in detailed genome-wide identifications and characterization of CaM and CML protein family, phylogenetic relationships, and domain structure. Expression study of CaM and CML genes were conducted in Glycine max and Phaseolus vulgaris . Our study provides a strong foundation for future functional research in CaM and CML gene family in plant kingdom.

Calmodulin (CaM) and calmodulin-like (CML) proteins are Ca 2+ relays and play diverse and multiple roles in plant growth, development and stress responses. However, CaM/CML gene family has not been identified in barley ( Hordeum vulgare ). In the present study, 5 HvCaMs and 80 HvCMLs were identified through a genome-wide analysis. All HvCaM proteins possessed 4 EF-hand motifs, whereas HvCMLs contained 1 to 4 EF-hand motifs. HvCaM2 , HvCaM3 and HvCaM5 coded the same polypeptide although they differed in nucleotide sequence, which was identical to the polypeptides coded by OsCaM1-1 , OsCaM1-2 and OsCaM1-3 . HvCaMs/CMLs were unevenly distributed over barley 7 chromosomes, and could be phylogenetically classified into 8 groups. HvCaMs/CMLs differed in gene structure, cis -acting elements and tissue expression patterns. Segmental and tandem duplication were observed among HvCaMs/CMLs during evolution. HvCML16 , HvCML18 , HvCML50 and HvCML78 were dispensable genes and the others were core genes in barley pan-genome. In addition, 14 HvCaM/CML genes were selected to examine their responses to salt, osmotic and low potassium stresses by qRT-PCR, and their expression were stress-and time-dependent. These results facilitate our understanding and further functional identification of HvCaMs/CMLs .