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• Genes encoding interacting proteins tend to be co-retained after whole-genome duplication (WGD). The preferential retention after WGD has been explained by the gene balance hypothesis (GBH). However, small-scale duplications could independently occur in the connected gene families. Certain evolutionary strategies might keep the dosage balanced. • Here, we examined the gene duplication, interaction and expression patterns of calcineurin B-like (CBL) and CBL-interacting protein kinase (CIPK) gene families to understand the underlying principles. • The ratio of the CBL and CIPK gene numbers evolved from 5 : 7 in Physcomitrella to 10 : 26 in Arabidopsis, and retrotransposition, tandem duplication, and WGDs contributed to the expansion. Two pairs of CBLs and six pairs of CIPKs were retained after the α WGD in Arabidopsis, in which specific interaction patterns were identified. In some cases, two retained CBLs (CIPKs) might compete to interact with a sole CIPK (CBL). Results of gene expression analyses indicated that the relatively over-retained duplicates tend to show asymmetric expression, thus avoiding competition. • In conclusion, our results suggested that the highly specific interaction, together with the differential gene expression pattern, jointly maintained the balanced dosage for the interacting CBL and CIPK proteins.

AP2C1 dephosphorylates CIPK9 to negatively regulate its function in controlling root growth and seedling development under low-K+ conditions in Arabidopsis. Abstract Potassium (K+) is a major macronutrient required for plant growth. An adaptive mechanism to low-K+ conditions involves activation of the Ca2+ signaling network that consists of calcineurin B-like proteins (CBLs) and CBL-interacting kinases (CIPKs). The CBL-interacting protein kinase 9 (CIPK9) has previously been implicated in low-K+ responses in Arabidopsis thaliana. Here, we report a protein phosphatase 2C (PP2C), AP2C1, that interacts with CIPK9. Fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC), and co-localization analyses revealed that CIPK9 and AP2C1 interact in the cytoplasm. AP2C1 dephosphorylates the auto-phosphorylated form of CIPK9 in vitro, presenting a regulatory mechanism for CIPK9 function. Furthermore, genetic and molecular analyses revealed that ap2c1 null mutants (ap2c1-1 and ap2c1-2) are tolerant to low-K+ conditions, retain higher K+ content, and show higher expression of K+-deficiency related genes contrary to cipk9 mutants (cipk9-1 and cipk9-2). In contrast, transgenic plants overexpressing AP2C1 were sensitive to low-K+ conditions. Thus, this study shows that AP2C1 and CIPK9 interact to regulate K+-deficiency responses in Arabidopsis. CIPK9 functions as positive regulator whereas AP2C1 acts as a negative regulator of Arabidopsis root growth and seedling development under low-K+ conditions.

Summary Wheat stripe rust caused by the fungus Puccinia striiformis f. sp. tritici (Pst) is one of the most destructive wheat diseases resulting in significant losses to wheat production worldwide. The development of disease‐resistant varieties is the most economical and effective measure to control diseases. Altering the susceptibility genes that promote pathogen compatibility via CRISPR/Cas9‐mediated gene editing technology has become a new strategy for developing disease‐resistant wheat varieties. Calcineurin B‐like protein (CBL)‐interacting protein kinases (CIPKs) has been demonstrated to be involved in defence responses during plant‐pathogen interactions. However, whether wheat CIPK functions as susceptibility factor is still unclear. Here, we isolated a CIPK homoeologue gene TaCIPK14 from wheat. Knockdown of TaCIPK14 significantly increased wheat resistance to Pst, whereas overexpression of TaCIPK14 resulted in enhanced wheat susceptibility to Pst by decreasing different aspects of the defence response, including accumulation of ROS and expression of pathogenesis‐relative genes. We generated wheat Tacipk14 mutant plants by simultaneous modification of the three homoeologues of wheat TaCIPK14 via CRISPR/Cas9 technology. The Tacipk14 mutant lines expressed race‐nonspecific (RNS) broad‐spectrum resistance (BSR) to Pst. Moreover, no significant difference was found in agronomic yield traits between Tacipk14 mutant plants and Fielder control plants under greenhouse and field conditions. These results demonstrate that TaCIPK14 acts as an important susceptibility factor in wheat response to Pst, and knockout of TaCIPK14 represents a powerful strategy for generating new disease‐resistant wheat varieties with BSR to Pst.

Abiotic stressors, including drought, salt, cold, and heat, profoundly impact plant growth and development, forcing elaborate cellular responses for adaptation and resilience. Among the crucial orchestrators of these responses is the CBL-CIPK pathway, comprising calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs). While CIPKs act as serine/threonine protein kinases, transmitting calcium signals, CBLs function as calcium sensors, influencing the plant’s response to abiotic stress. This review explores the intricate interactions between the CBL-CIPK pathway and plant hormones such as ABA, auxin, ethylene, and jasmonic acid (JA). It highlights their role in fine-tuning stress responses for optimal survival and acclimatization. Building on previous studies that demonstrated the enhanced stress tolerance achieved by upregulating CBL and CIPK genes, we explore the regulatory mechanisms involving post-translational modifications and protein–protein interactions. Despite significant contributions from prior research, gaps persist in understanding the nuanced interplay between the CBL-CIPK system and plant hormone signaling under diverse abiotic stress conditions. In contrast to broader perspectives, our review focuses on the interaction of the pathway with crucial plant hormones and its implications for genetic engineering interventions to enhance crop stress resilience. This specialized perspective aims to contribute novel insights to advance our understanding of the potential of the CBL-CIPK pathway to mitigate crops’ abiotic stress.

Calcium serves as a versatile messenger in many adaptation and developmental processes in plants. Cellular calcium signals are detected and transmitted by calcium-binding proteins functioning as sensor molecules. The family of calcineurin B-like (CBL) proteins represents a unique group of calcium sensors and contributes to the decoding of calcium transients by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). In higher plants, CBL proteins and CIPKs form a complex signaling network that allows for flexible but specific signal-response coupling during environmental adaptation reactions. This review presents novel findings concerning the evolution of this signaling network and key insights into the physiological function of CBL-CIPK complexes. These aspects will be presented and discussed in the context of emerging functional principles governing efficient and specific information processing in this signaling system.

The calcium sensor TaCBL4 and its interacting kinase protein TaCIPK5 positively contribute to wheat resistance to Puccinia striiformis f. sp. tritici in a ROS-dependent manner. Abstract Calcineurin B-like proteins (CBLs) act as Ca2+ sensors to activate specific protein kinases, namely CBL-interacting protein kinases (CIPKs). Recent research has demonstrated that the CBL-CIPK complex is not only required for abiotic stress signaling, but is also probably involved in biotic stress perception. However, the role of this complex in immune signaling, including pathogen perception, is unknown. In this study, we isolated one signaling component of the TaCBL-TaCIPK complex (TaCBL4-TaCIPK5) and characterized its role in the interaction between wheat (Triticum aestivum) and Puccinia striiformis f. sp. tritici (Pst, stripe rust fungus). Among all TaCBLs in wheat, TaCBL4 mRNA accumulation markedly increased after infection by Pst. Silencing of TaCBL4 resulted in enhanced susceptibility to avirulent Pst infection. In addition, screening determined that TaCIPK5 physically interacted with TaCBL4 in planta and positively contributed to wheat resistance to Pst. Moreover, the disease resistance phenotype of TaCBL4 and TaCIPK5 co-silenced plants was consistent with that of single-knockdown plants. The accumulation of reactive oxygen species (ROS) was significantly altered in all silenced plants during Pst infection. Together these findings demonstrate that the TaCBL4-TaCIPK5 complex positively modulates wheat resistance in a ROS-dependent manner, and provide new insights into the roles of CBL-CIPK in wheat.

Background Calcineurin B-like proteins (CBLs) are ubiquitous Ca 2+ sensors that mediate plant responses to various stress and developmental processes by interacting with CBL-interacting protein kinases (CIPKs). CBLs and CIPKs play essential roles in acclimatization of crop plants. However, evolution of these two gene families in the genus Medicago is poorly understood. Results A total of 68 CBL and 135 CIPK genes have been identified in five genomes from Medicago . Among these genomes, the gene number of CBL s and CIPK s shows no significant difference at the haploid genome level. Phylogenetic and comprehensive characteristic analyses reveal that CBL s and CIPK s are classified into four clades respectively, which is validated by distribution of conserved motifs. The synteny analysis indicates that the whole genome duplication events (WGDs) have contributed to the expansion of both families. Expression analysis demonstrates that two MsCBL s and three MsCIPK s are specifically expressed in roots, mature leaves, developing flowers and nitrogen fixing nodules of Medicago sativa spp. sativa , the widely grown tetraploid species. In particular, the expression of these five genes was highly up-regulated in roots when exposed to salt and drought stress, indicating crucial roles in stress responses. Conclusions Our study leads to a comprehensive understanding of evolution of CBL and CIPK gene families in Medicago , but also provides a rich resource to further address the functions of CBL-CIPK complexes in cultivated species and their closely related wild relatives.

Calcium serves as a versatile messenger in many adaptation and developmental processes in plants. Cellular calcium signals are detected and transmitted by calcium-binding proteins functioning as sensor molecules. The family of calcineurin B-like (CBL) proteins represents a unique group of calcium sensors and contributes to the decoding of calcium transients by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). In higher plants, CBL proteins and CIPKs form a complex signaling network that allows for flexible but specific signal–response coupling during environmental adaptation reactions. This review presents novel findings concerning the evolution of this signaling network and key insights into the physiological function of CBL–CIPK complexes. These aspects will be presented and discussed in the context of emerging functional principles governing efficient and specific information processing in this signaling system.

Context Calcium-dependent signaling in plants is responsible for several major cellular events, including the activation of the salinity-responsive pathways. Calcium binds to calcineurin B-like protein (CBL), and the resulting CBL-Ca 2+ complex binds to CBL-interacting protein kinase (CIPK). The CBL-CIPK complex enhances the CIPK interaction with an upstream kinase. The upstream kinase phosphorylates CIPK that, in turn, phosphorylates membrane transporters. Phosphorylation influences transporter activity to kick-start many downstream functions, such as balancing the cytosolic Na + -to-K + ratio. The CBL-CIPK interaction is pivotal for Ca 2+ -dependent salinity stress signaling. Methods Computational methods are used to model the entire Arabidopsis thaliana CIPK24 protein structure in its autoinhibited and open-activated states. Arabidopsis thaliana CIPK24-CBL4 complex is predicted based on the protein–protein docking methods. The available structural and functional data support the CIPK24 and the CIPK24-CBL4 complex models. Models are energy-minimized and subjected to molecular dynamics (MD) simulations. MD simulations for 500 ns and 300 ns enabled us to predict the importance of conserved residues of the proteins. Finally, the work is extended to predict the CIPK24-CBL4 complex with the upstream kinase GRIK2. MD simulation for 300 ns on the ternary complex structure enabled us to identify the critical CIPK24-GRIK2 interactions. Together, these data could be used to engineer the CBL-CIPK interaction network for developing salt tolerance in crops.

Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 ( ) and 26 ( ) genes were isolated from cassava by genome searching and cloning of cDNA sequences of s and s. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of and genes were different in different tissues throughout the life cycle. The expression patterns of 7 and 26 genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR), and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava , and Na /H antiporter genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses.