CBL-interacting Protein Kinases Gene Research Articles

Molecular Mechanisms of CBL-CIPK Signaling Pathway in Plant Abiotic Stress Tolerance and Hormone Crosstalk.

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.

Genome-wide identification, expression analysis, and abiotic stress response of the CBL and CIPK gene families in Artocarpus nanchuanensis

Artocarpus nanchuanensis, the northernmost species in the jackfruit genus, has great economic and horticultural value due to its nutritious fruit and beautiful tree shape. Calcineurin B-like proteins (CBLs) act as plant-specific Ca2+ sensors and participate in regulating plant responses to various abiotic stresses by interacting with CBL-interacting protein kinases (CIPKs). However, the characteristics and functions of the CBL and CIPK genes in A. nanchuanensis are still unclear. Here, we identified 14 CBL and 33 CIPK genes from the A. nanchuanensis genome, and based on phylogenetic analysis, they were divided into 4 and 7 clades, respectively. Gene structure and motif analysis indicated that the AnCBL and AnCIPK genes were relatively conserved. Colinear analysis showed that segmental duplication contributed to the expansion of the AnCBL and AnCIPK gene families. Expression analysis showed that AnCBL and AnCIPK genes were widely expressed in various tissues of A. nanchuanensis and exhibited tissue-specific expression. In addition, three genes (AnCBL6, AnCIPK7/8) may play important roles in response to salt, cold, and drought stresses. In summary, this study lays an important foundation for the improvement of stress resistance in A. nanchuanensis and provides new insight for the functional research on CBL and CIPK gene families.

Understanding the salt overly sensitive pathway in Prunus: Identification and characterization of NHX, CIPK, and CBL genes.

Salinity is a major abiotic stress factor that can significantly impact crop growth, and productivity. In response to salt stress, the plant Salt Overly Sensitive (SOS) signaling pathway regulates the homeostasis of intracellular sodium ion concentration. The SOS1, SOS2, and SOS3 genes play critical roles in the SOS pathway, which belongs to the members of Na+/H+ exchanger (NHX), CBL-interacting protein kinase (CIPK), and calcineurin B-like (CBL) gene families, respectively. In this study, we performed genome-wide identifications and phylogenetic analyses of NHX, CIPK, and CBL genes in six Rosaceae species: Prunus persica, Prunus dulcis, Prunus mume, Prunus armeniaca, Pyrus ussuriensis × Pyrus communis, and Rosa chinensis. NHX, CIPK, and CBL genes of Arabidopsis thaliana were used as controls for phylogenetic analyses. Our analysis revealed the lineage-specific and adaptive evolutions of Rosaceae genes. Our observations indicated the existence of two primary classes of CIPK genes: those that are intron-rich and those that are intron-less. Intron-rich CIPKs in Rosaceae and Arabidopsis can be traced back to algae CIPKs and CIPKs found in early plants, suggesting that intron-less CIPKs evolved from their intron-rich counterparts. This study identified one gene for each member of the SOS signaling pathway in P. persica: PpSOS1, PpSOS2, and PpSOS3. Gene expression analyses indicated that all three genes of P. persica were expressed in roots and leaves. Yeast two-hybrid-based protein-protein interaction analyses revealed a direct interaction between PpSOS3 and PpSOS2; and between PpSOS2 and PpSOS1C-terminus region. Our findings indicate that the SOS signaling pathway is highly conserved in P. persica.

Identification of CBL and CIPK Gene Families and Functional Characterization of PvCIPK7 as an Essential Regulator of Root Nodule Development and Nitrogen Fixation in Phaseolus vulgaris

Calcineurin B-like proteins (CBLs) act as Ca2+ sensors and activate CBL-interacting protein kinases (CIPKs) to form CBL-CIPK complexes, crucial for plant signal transduction and stress perception. Nevertheless, their functions in root nodule symbiosis (RNS) are largely unexplored. Herein, we classified previously identified SnRK3s as CIPKs and identified 10 CBLs in Phaseolus vulgaris. Additionally, we conducted a functional characterization of PvCIPK7 to acquire a comprehensive understanding of its involvement in RNS in P. vulgaris. Transcript downregulation of PvCIPK7 by RNA interference (RNAi) significantly affected primary and lateral root growth and density. During symbiosis, PvCIPK7-RNAi roots showed a reduction in infection events, nodule numbers, and, nodule size was significantly reduced. The cross section of PvCIPK7-RNAi nodule revealed a loss of nodule organization, a reduction in the number of symbiosomes in infected cells, and a failure of Rhizobium bacteria to differentiate into bacteroids. This severely affected the nitrogen-fixing ability of PvCIPK7-RNAi nodules, as evidenced by downregulated symbiosis-specific genes and genes related to nitrogen fixation. The spatiotemporal expression pattern of the CIPK7 promoter revealed symbiosis-associated expression in the early stages. In conclusion, PvCIPK7 is indispensable for root nodule development, bacterial differentiation, and, consequently, nitrogen fixation during the symbiotic interaction between rhizobia and P. vulgaris.

The Effect of Stress Hormones, Ultraviolet C, and Stilbene Precursors on Expression of Calcineurin B-like Protein (CBL) and CBL-Interacting Protein Kinase (CIPK) Genes in Cell Cultures and Leaves of Vitis amurensis Rupr

Calcium serves as a crucial messenger in plant stress adaptation and developmental processes. Plants encode several multigene families of calcium sensor proteins with diverse functions in plant growth and stress responses. Several studies indicated that some calcium sensors may be involved in the regulation of secondary metabolite production in plant cells. The present study aimed to investigate expression of calcineurin B-like proteins (CBL) and CBL-interacting protein kinase (CIPK) in response to conditions inducting biosynthesis of stilbenes in grapevine. We investigated CBL and CIPK gene expression in wild-growing grapevine Vitis amurensis Rupr., known as a rich stilbene source, in response to the application of stilbene biosynthesis-inducing conditions, including application of stress hormones (salicylic acid or SA, methyl jasmonate or MeJA), phenolic precursors (p-coumaric acids or CA), and ultraviolet irradiation (UV-C). The influence of these effectors on the levels of 13 VaCBL and 27 VaCIPK mRNA transcripts as well as on stilbene production was analyzed by quantitative real-time RT-PCR in the leaves and cell cultures of V. amurensis. The data revealed that VaCBL4-1 expression considerably increased after UV-C treatment in both grapevine cell cultures and leaves. The expression of VaCIPK31, 41-1, and 41-2 also increased, but this increase was mostly detected in cell cultures of V. amurensis. At the same time, expression of most VaCBL and VaCIPK genes was markedly down-regulated both in leaves and cell cultures of V. amurensis, which may indicate that the CBLs and CIPKs are involved in negative regulation of stilbene accumulation (VaCBL8, 10a-2, 10a-4, 11, 12, VaCIPK3, 9-1, 9-2, 12, 21-1, 21-2, 33, 34, 35, 36, 37, 39, 40, 41-3, 41-4). The results obtained provide new information of CBL and CIPK implication in the regulation of plant secondary metabolism in response to stress hormones, metabolite precursors, and UV-C irradiation.

AtCIPK16, a CBL-interacting protein kinase gene, confers salinity tolerance in transgenic wheat.

Globally, wheat is the major source of staple food, protein, and basic calories for most of the human population. Strategies must be adopted for sustainable wheat crop production to fill the ever-increasing food demand. Salinity is one of the major abiotic stresses involved in plant growth retardation and grain yield reduction. In plants, calcineurin-B-like proteins form a complicated network with the target kinase CBL-interacting protein kinases (CIPKs) in response to intracellular calcium signaling as a consequence of abiotic stresses. The AtCIPK16 gene has been identified in Arabidopsis thaliana and found to be significantly upregulated under salinity stress. In this study, the AtCIPK16 gene was cloned in two different plant expression vectors, i.e., pTOOL37 having a UBI1 promoter and pMDC32 having a 2XCaMV35S constitutive promoter transformed through the Agrobacterium-mediated transformation protocol, in the local wheat cultivar Faisalabad-2008. Based on their ability to tolerate different levels of salt stress (0, 50, 100, and 200 mM), the transgenic wheat lines OE1, OE2, and OE3 expressing AtCIPK16 under the UBI1 promoter and OE5, OE6, and OE7 expressing the same gene under the 2XCaMV35S promoter performed better at 100 mM of salinity stress as compared with the wild type. The AtCIPK16 overexpressing transgenic wheat lines were further investigated for their K+ retention ability in root tissues by utilizing the microelectrode ion flux estimation technique. It has been demonstrated that after 10min of 100 mM NaCl application, more K+ ions were retained in the AtCIPK16 overexpressing transgenic wheat lines than in the wild type. Moreover, it could be concluded that AtCIPK16 functions as a positive elicitor in sequestering Na+ ions into the cell vacuole and retaining more cellular K+ under salt stress to maintain ionic homeostasis.

Transcriptome-Wide Identification and Functional Characterization of CIPK Gene Family Members in Actinidia valvata under Salt Stress

Fruit plants are severely constrained by salt stress in the soil due to their sessile nature. Ca2+ sensors, which are known as CBL-interacting protein kinases (CIPKs), transmit abiotic stress signals to plants. Therefore, it is imperative to investigate the molecular regulatory role of CIPKs underlying salt stress tolerance in kiwifruit. In the current study, we have identified 42 CIPK genes from Actinidia. valvata (A.valvata). All the AvCIPKs were divided into four different phylogenetic groups. Moreover, these genes showed different conserved motifs. The expression pattern analysis showed that AvCIPK11 was specifically highly expressed under salt stress. The overexpression of AvCIPK11 in 'Hongyang' (a salt sensitive commercial cultivar from Actinidia chinensis) enhanced salt tolerance by maintaining K+/Na+ homeostasis in the leaf and positively improving the activity of POD. In addition, the salt-related genes AcCBL1 and AcNHX1 had higher expression in overexpression lines. Collectively, our study suggested that AvCIPK11 is involved in the positive regulation of salt tolerance in kiwifruit.

Genome Wide Characterization of CBL-CIPK Family Genes and Their Responsive Expression in Rosa chinensis

Calcium (Ca2+) plays a pivotal role in various signal transduction pathways. Calcineurin B-like proteins (CBLs) are a unique group of Ca2+ sensors that decode Ca2+ signals by activating the plant specific protein kinase known as the CBL-interacting protein kinase (CIPK). In plants, the CBL-CIPK signaling network regulates multiple signals in response to different extracellular cues including abiotic stress. However, the genome wide annotation and expression patterns of CBLs and CIPKs in woody cutting flower plants are still unclear. In this study, a total number of 7 CBLs (RcCBLs) and 17 CIPKs (RcCIPKs) genes, divided into four and five subfamilies, respectively, were identified from the rose genome. All RcCBLs possess a classic elongation factor-hand (EF-hand) domain, while all RcCIPKs possess both the classic kinase and NAF domains. Most RcCBLs were predicted to be plasma membrane localized, whereas most RcCIPKs were predicted to be cytoplasmic localized. Synteny analysis showed that one RcCBL gene pair and five RcCIPK gene pairs have gone through whole genome duplication events. Promoter cis-element prediction assays indicated that RcCBLs and RcCIPKs could function in different abiotic stress responses in rose plants. Further quantitative real-time PCR analysis demonstrated that RcCBLs and RcCIPKs were expressed in different organs with overlapped but distinct patterns in response to various abiotic stresses. The findings in this work will provide fundamental information and gene resources for further functional research on RcCBLs and RcCIPKs.

Evolution of the CBL and CIPK gene families in Medicago: genome-wide characterization, pervasive duplication, and expression pattern under salt and drought stress

BackgroundCalcineurin B-like proteins (CBLs) are ubiquitous Ca2+ 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.ResultsA total of 68 CBL and 135 CIPK genes have been identified in five genomes from Medicago. Among these genomes, the gene number of CBLs and CIPKs shows no significant difference at the haploid genome level. Phylogenetic and comprehensive characteristic analyses reveal that CBLs and CIPKs 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 MsCBLs and three MsCIPKs 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.ConclusionsOur 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.

Grape CIPK18 acts as a positive regulator of CBF cold signaling pathway by modulating ROS homeostasis

Cold temperatures trigger a diverged Ca 2+ decoding paradigm that includes the proteins CBL (Calcineurin B-like proteins) and CIPK (CBL-interacting protein kinases). Here, putative CBL and CIPK genes were surveyed in cold-tolerant wild Amur grape ( Vitis amurensis ), designated as VaCBLs and VaCIPKs , respectively. The transcripts of 8 VaCBLs and 19 VaCIPKs encoded in this genome were confirmed, among which VaCIPK18 physically interacted with all 8 VaCBLs through the C-terminal regulatory domain (NAF), indicating that it may serve as a node regulator. VaCIPK18 was ubiquitously expressed in various tissues and was highly induced by cold, drought, salt, and ABA . The NAF domain of VaCIPK18 was required for functional homodimerization in yeast as well as in planta . Complementation tests confirmed that VaCIPK18 rescued the cold sensitivity of ArabidopsisAtcipk3 mutant, in which the expressions of CBFs were suppressed and is sensitive to cold and freezing stresses. In addition, overexpression of VaCIPK18 enhanced the cold tolerance by upregulating the C-repeat binding transcription factor (CBF) transcriptional pathway under normal or cold conditions, and reducing reactive oxygen species production. Thus, our study demonstrates that VaCIPK18 positively regulates cold signaling at least partly through the direct transcriptional control of cold-regulated CBFs and modulation of ROS homeostasis . Moreover, VaCIPK18 was found to interact with VaMYB4a in vivo and in vitro . Our findings provide evidence that VaCIPK18 functions as a key node in the CBL-CIPK network and may interact with VaMYB4a to contribute to cold response in grape plants. • An Amur grape VaCIPK18 could function as a node regulator of CBL-CIPK network. • VaCIPK18 modulates cold tolerance by directly controlling CBFs and ROS homeostasis. • 'VaCIPK18-VaMYB4a' module may regulate cold tolerance.

Molecular and expression analysis indicate the role of CBL interacting protein kinases (CIPKs) in abiotic stress signaling and development in chickpea

Calcineurin B-like proteins (CBL)-interacting protein kinases (CIPKs) regulate the developmental processes, hormone signal transduction and stress responses in plants. Although the genome sequence of chickpea is available, information related to the CIPK gene family is missing in this important crop plant. Here, a total of 22 CIPK genes were identified and characterized in chickpea. We found a high degree of structural and evolutionary conservation in the chickpea CIPK family. Our analysis showed that chickpea CIPKs have evolved with dicots such as Arabidopsis and soybean, and extensive gene duplication events have played an important role in the evolution and expansion of the CIPK gene family in chickpea. The three-dimensional structure of chickpea CIPKs was described by protein homology modelling. Most CIPK proteins are localized in the cytoplasm and nucleus, as predicted by subcellular localization analysis. Promoter analysis revealed various cis-regulatory elements related to plant development, hormone signaling, and abiotic stresses. RNA-seq expression analysis indicated that CIPKs are significantly expressed through a spectrum of developmental stages, tissue/organs that hinted at their important role in plant development. The qRT-PCR analysis revealed that several CaCIPK genes had specific and overlapping expressions in different abiotic stresses like drought, salt, and ABA, suggesting the important role of this gene family in abiotic stress signaling in chickpea. Thus, this study provides an avenue for detailed functional characterization of the CIPK gene family in chickpea and other legume crops.

Evolution, gene expression, and protein‒protein interaction analyses identify candidate CBL-CIPK signalling networks implicated in stress responses to cold and bacterial infection in citrus

BackgroundCold is a major abiotic stress and Huanglongbing and citrus canker disease are two devastating bacterial diseases for citrus. The Ca2+-CBL-CIPK network is known to regulate different types of stress signalling in plants. How do CBL–CIPK signalling networks function in response to cold and infection by CLas or Xcc in citrus?ResultsEight calcineurin B-like proteins (CBLs) and seventeen CBL-interacting protein kinases (CIPKs) were identified from the cold-tolerant satsuma mandarin ‘Guijing2501’ (Citrus. unshiu) and CLas/Xcc-sensitive sweet orange (C. sinensis). Phylogenetic analysis revealed that both CBL and CIPK family members in citrus were classified into an ancient and a recent clade according to their conserved domain characteristics and/or intron/exon structures. Genome duplication analysis suggested that both tandem and segmental duplications contributed to the amplification of the CBL and CIPK gene families in citrus under intense purifying selection, and the duplication events only existed in the recent clades. Expression comparison of the duplicated gene pairs indicated that the duplicated CBL and CIPK genes underwent functional differentiation. Further expression analysis identified that CBL1, 5, 6, and 8 and CIPK2, 8, 12, 15, 16, and 17 were significantly regulated by multiple stresses, including cold, Xcc infection and/or CLas infection, in citrus, whereas CBL2/7 and CIPK1/4/5/11/13/14 were independently highly regulated by cold and CIPK3 was uniquely responsive to Xcc infection. The combination analyses of targeted Y2H assay and expression analysis revealed that CBL6-CIPK8 was the common signalling network in response to cold and Xcc infection, while CBL6/CBL8-CIPK14 was uniquely responsive to cold in citrus. Further stable transformation and cold tolerance assay indicated that overexpression of CuCIPK16 enhanced the cold tolerance of transgenic Arabidopsis with higher POD activity and lower MDA content.ConclusionsIn this study, evolution, gene expression and protein‒protein interaction analyses of citrus CBLs and CIPKs were comprehensively conducted over a genome-wide range. The results will facilitate future functional characterization of individual citrus CBLs and CIPKs under specific stresses and provide clues for the clarification of cold tolerance and disease susceptibility mechanisms in corresponding citrus cultivars.

Genome-Wide Identification of the Salvia miltiorrhiza SmCIPK Gene Family and Revealing the Salt Resistance Characteristic of SmCIPK13

Members of the CIPK (CBL-interacting protein kinases) gene family play important roles in calcium (Ca2+) signaling pathway-regulated plant resistance to abiotic stresses. Salvia miltiorrhiza, which is widely planted and grown in complex and diverse environments, is mainly focused on the transcriptional regulation of enzyme genes related to the biosynthesis of its bioactive components. However, the excavation of the genes related to the resistance of S.miltiorrhiza and the involved signaling pathways have not been deeply studied. In this study, 20 SmCIPK genes were identified and classified into two families and five subfamilies by biochemical means. Sequence characteristics and conserved motif analysis revealed the conservation and difference of SmCIPK protein in plants. Expression pattern analysis showed that SmCIPKs were mainly expressed in flowers and roots, and more than 90% of gene expression was induced by SA (salicylic acid), and MeJA (methyl jasmonate). Furthermore, the expression level of SmCIPK13 could be significantly increased after stress treatment with NaCl. SmCIPK13 expression in yeast reduces sensitivity to salt, while overexpression of it in Arabidopsis has the same effect and was localized in the cytoplasm, cell membrane and nucleus. In conclusion, the identification of the SmCIPK gene family and the functional characterization of the SmCIPK13 gene provides the basis for clarification of key genes in the Ca2+ signaling pathway and abiotic stress in S.miltiorrhiza.

Identification of the CIPK-CBL family gene and functional characterization of CqCIPK14 gene under drought stress in quinoa

BackgroundCalcineurin-like Protein (CBL) and CBL interacting protein kinase (CIPK) play a key role in plant signal transduction and response to various environmental stimuli. Quinoa, as an important plant with high nutritional value, can meet the basic nutritional needs of human Cash crop, is also susceptible to abiotic stress. However, CBL-CIPK in quinoa have not been reported.ResultsIn this study, 16 CBL and 41 CIPK genes were identified in quinoa. CBL-CIPK gene shows different intron-exon gene structure and motif, they participate in different biological processes, and form a complex regulatory network between CBL-CIPK proteins. Many cis-regulatory element associated with ABA and drought have been found. The expression patterns of CBL-CIPK showed different expression patterns in various abiotic stresses and tissues. RT-qPCR showed that most members of these two gene families were involved in drought regulation of quinoa, in particular, the expression levels of CqCIPK11, CqCIPK15, CqCIPK37 and CqCBL13 increased significantly under drought stress.ConclusionsThe structures and functions of the CBL-CIPK family in quinoa were systematically explored. Many CBL-CIPK may play vital roles in the regulation of organ development, growth, and responses to abiotic stresses. This research has great significance for the functional characterisation of the quinoa CBL-CIPK family and our understanding of the CBL-CIPK family in higher plants.

Insight into the CBL and CIPK gene families in pecan (Carya illinoinensis): identification, evolution and expression patterns in drought response

BackgroundCalcium (Ca2+) serves as a ubiquitous second messenger and plays a pivotal role in signal transduction. Calcineurin B-like proteins (CBLs) are plant-specific Ca2+ sensors that interact with CBL-interacting protein kinases (CIPKs) to transmit Ca2+ signals. CBL-CIPK complexes have been reported to play pivotal roles in plant development and response to drought stress; however, limited information is available about the CBL and CIPK genes in pecan, an important nut crop.ResultsIn the present study, a total of 9 CBL and 30 CIPK genes were identified from the pecan genome and divided into four and five clades based on phylogeny, respectively. Gene structure and distribution of conserved sequence motif analysis suggested that family members in the same clade commonly exhibited similar exon-intron structures and motif compositions. The segmental duplication events contributed largely to the expansion of pecan CBL and CIPK gene families, and Ka/Ks values revealed that all of them experienced strong negative selection. Phylogenetic analysis of CIPK proteins from 14 plant species revealed that CIPKs in the intron-poor clade originated in seed plants. Tissue-specific expression profiles of CiCBLs and CiCIPKs were analysed, presenting functional diversity. Expression profiles derived from RNA-Seq revealed distinct expression patterns of CiCBLs and CiCIPKs under drought treatment in pecan. Moreover, coexpression network analysis helped to elucidate the relationships between these genes and identify potential candidates for the regulation of drought response, which were verified by qRT–PCR analysis.ConclusionsThe characterization and analysis of CBL and CIPK genes in pecan genome could provide a basis for further functional analysis of CiCBLs and CiCIPKs in the drought stress response of pecan.

A CBL-interacting protein kinase, AcCIPK18, from Ananas comosus regulates tolerance to salt, drought, heat stress and Sclerotinia sclerotiorum infection in Arabidopsis

In plants, calcineurin B-like proteins (CBLs) and their interacting kinase, CBL-interacting protein kinases (CIPKs), perform essential roles in mediating responses to various extracellular cues, including biotic and abiotic stresses. Several CIPK genes have been functionally characterized in different plant species. However, to date, the functions of pineapple CIPKs have not been investigated, which limits their potential use in molecular breeding. Here we functionally characterized an AcCIPK18 of pineapple and verified its role in salt, drought, heat stresses and immunity. Bioinformatics studies and phylogenetic analysis showed that AcCIPK18 is homologous to CIPK6 of Arabidopsis. Expression analysis shows that AcCIPK18 gets induced by salt, osmotic, heat and ABA. Besides, the ectopic expression of AcCIPK18 increased tolerance to salt, drought and heat stress but displayed sensitivity against fungal infection in Arabidopsis. We found that overexpression of AcCIPK18 changes the ROS homeostasis by altering the expression of several ABA signaling genes. These results provide evidence for the involvement of AcCIPK18 in multiple stress responses and advocates that AcCIPK18 could be used as a potential candidate gene for improving tolerance against multiple abiotic stress.

Genome-Wide Identification, Characterization and Expression Analysis of the CIPK Gene Family in Potato (Solanum tuberosum L.) and the Role of StCIPK10 in Response to Drought and Osmotic Stress.

The potato (Solanum tuberosum L.), one of the most important food crops worldwide, is sensitive to environmental stresses. Sensor–responder complexes comprising calcineurin B-like (CBL) proteins and CBL-interacting protein kinases (CIPKs) not only modulate plant growth and development but also mediate numerous stress responses. Here, using a Hidden Markov Model and BLAST searches, 27 CIPK genes were identified in potato and divided into five groups by phylogenetic analysis and into two clades (intron-poor and intron-rich) by gene structure analysis. Quantitative reverse-transcription PCR (qRT-PCR) assays revealed that StCIPK genes play important roles in plant growth, development and abiotic stress tolerance. Up-regulated expression of StCIPK10 was significantly induced by drought, PEG6000 and ABA. StCIPK10 enhances both the ability of potato to scavenge reactive oxygen species and the content of corresponding osmoregulation substances, thereby strengthening tolerance to drought and osmotic stress. StCIPK10 is located at the intersection between the abscisic acid and abiotic stress signaling pathways, which control both root growth and stomatal closure in potato. In addition, StCIPK10 interacts with StCBL1, StCBL4, StCBL6, StCBL7, StCBL8, StCBL11 and StCBL12, and is specifically recruited to the plasma membrane by StCBL11.

The CBL-interacting protein kinase CaCIPK13 positively regulates defence mechanisms against cold stress in pepper.

Cold stress is one of the main factors limiting growth and development in pepper. Calcineurin B-like proteins (CBLs) are specific calcium sensors with non-canonical EF-hands to capture calcium signals, and interact with CBL-interacting protein kinases (CIPKs) in the regulation of various stresses. In this study, we isolated a cold-induced CIPK gene from pepper named CaCIPK13, which encodes a protein of 487 amino acids. In silico analyses indicated that CaCIPK13 is a typical CIPK family member with a conserved NAF motif, which consists of the amino acids asparagine, alanine, and phenylalanine. The CaCIPK13 protein was located in the nucleus and plasma membrane. Knock down of CaCIPK13 resulted in enhanced sensitivity to cold stress in pepper, with increased malondialdehyde content, H2O2 accumulation, and electrolyte leakage, while the catalase, peroxidase, superoxide dismutase activities and anthocyanin content were decreased. The transcript level of cold and anthocyanin-related genes was substantially decreased in CaCIPK13-silenced pepper leaves relative to the empty vector control. On the contrary, overexpression of CaCIPK13 in tomato improved cold tolerance via increasing anthocyanin content and activities of reactive oxygen species scavenging enzymes. Furthermore, the interaction of CaCIPK13 with CaCBL1/6/7/8 was Ca2+-dependent. These results indicate that CaCIPK13 plays a positive role in cold tolerance mechanism via CBL-CIPK signalling.

Genome-Wide Identification of CBL-CIPK Gene Family in Honeysuckle (Lonicera japonica Thunb.) and Their Regulated Expression Under Salt Stress.

In plants, calcineurin B-like proteins (CBLs) are a unique group of Ca2+ sensors that decode Ca2+ signals by activating a family of plant-specific protein kinases known as CBL-interacting protein kinases (CIPKs). CBL-CIPK gene families and their interacting complexes are involved in regulating plant responses to various environmental stimuli. To gain insight into the functional divergence of CBL-CIPK genes in honeysuckle, a total of six LjCBL and 17 LjCIPK genes were identified. The phylogenetic analysis along with the gene structure analysis divided both CBL and CBL-interacting protein kinase genes into four subgroups and validated by the distribution of conserved protein motifs. The 3-D structure prediction of proteins shown that most LjCBLs shared the same Protein Data Bank hit 1uhnA and most LjCIPKs shared the 6c9Da. Analysis of cis-acting elements and gene ontology implied that both LjCBL and LjCIPK genes could be involved in hormone signal responsiveness and stress adaptation. Protein-protein interaction prediction suggested that LjCBL4 is hypothesized to interact with LjCIPK7/9/15/16 and SOS1/NHX1. Gene expression analysis in response to salinity stress revealed that LjCBL2/4, LjCIPK1/15/17 under all treatments gradually increased over time until peak expression at 72 h. These results demonstrated the conservation of salt overly sensitive pathway genes in honeysuckle and a model of Ca2+-LjCBL4/LjSOS3-LjCIPK16/LjSOS2 module-mediated salt stress signaling in honeysuckle is proposed. This study provides insight into the characteristics of the CBL-CIPK gene families involved in honeysuckle salt stress responses, which could serve as a foundation for gene transformation technology, to obtain highly salt-tolerant medicinal plants in the context of the global reduction of cultivated land.

The pigeon pea CcCIPK14-CcCBL1 pair positively modulates drought tolerance by enhancing flavonoid biosynthesis.

Calcineurin B-like (CBL)-interacting protein kinases (CIPKs) play a central role in Ca2+ signalling and promote drought tolerance in plants. The CIPK gene family in pigeon pea (Cajanus cajan L.), a major food crop affected by drought, has not previously been characterised. Here, we identified 28 CIPK genes in the pigeon pea genome. Five CcCIPK genes were strongly upregulated in roots upon drought treatment and were selected for further characterisation. Overexpression of CcCIPK13 and CcCIPK14 increased survival rates by two- to three-fold relative to controls after 14days of drought. Furthermore, the three major flavonoids, genistin, genistein and apigenin, were significantly upregulated in the same transgenic plants. Using CcCIPK14 as bait, we performed a yeast two-hybrid screen and identified six interactors, including CcCBL1. CcCIPK14 exhibited autophosphorylation and phosphorylation of CcCBL1 in vitro. CcCBL1-overexpressed plants displayed higher survival rates upon drought stress as well as higher expression of flavonoid biosynthetic genes and flavonoid content. CcCIPK14-overexpressed plants in which CcCBL1 transcript levels were reduced by RNA interference had lower survival rates, which indicated CcCBL1 in the same pathway as CcCIPK14. Together, our results demonstrate a role for the CcCIPK14-CcCBL1 complex in drought stress tolerance through the regulation of flavonoid biosynthesis in pigeon pea.