Potassium (K+) plays a role in enzyme activation, membrane transport, and osmotic regulation processes. An increase in potassium content can significantly improve the elasticity and combustibility of tobacco and reduce the content of harmful substances. Here, we report that the expression analysis of Nt GF14e, a 14-3-3 gene, increased markedly after low-potassium treatment (LK). Then, chlorophyll content, POD activity and potassium content, were significantly increased in overexpression of Nt GF14e transgenic tobacco lines compared with those in the wild type plants. The net K+ efflux rates were severely lower in the transgenic plants than in the wild type under LK stress. Furthermore, transcriptome analysis identified 5708 upregulated genes and 2787 downregulated genes between Nt GF14e overexpressing transgenic tobacco plants. The expression levels of some potassium-related genes were increased, such as CBL-interacting protein kinase 2 (CIPK2), Nt CIPK23, Nt CIPK25, H+-ATPase isoform 2 a (AHA2a), Nt AHA4a, Stelar K+ outward rectifier 1(SKOR1), and high affinity K+ transporter 5 (HAK5). The result of yeast two-hybrid and luciferase complementation imaging experiments suggested Nt GF14e could interact with CIPK2. Overall, these findings indicate that NtGF14e plays a vital roles in improving tobacco LK tolerance and enhancing potassium nutrition signaling pathways in tobacco plants.

Mg-K homeostasis is essential for plant response to abiotic stress, but its regulation remains largely unknown. MsWRKY44 cloned from alfalfa was highly expressed in leaves and petioles. Overexpression of it inhibited alfalfa growth, and promoted leaf senescence and alfalfa sensitivities to acid and Al stresses. The leaf tips, margins and interveins of old leaves occurred yellow spots in MsWRKY44-OE plants under pH4.5 and pH4.5 +Al conditions. Meanwhile, Mg-K homeostasis was substantially changed with reduction of K accumulation and increases of Mg as well as Al accumulation in shoots of MsWRKY44-OE plants. Further, MsWRKY44 was found to directly bind to the promoters of MsMGT7 and MsCIPK23, and positively activated their expression. Transiently overexpressed MsMGT7 and MsCIPK23 in tobacco leaves increased the Mg and Al accumulations but decreased K accumulation. These results revealed a novel regulatory module MsWRKY44-MsMGT7/MsCIPK23, which affects the transport and accumulation of Mg and K in shoots, and promotes alfalfa sensitivities to acid and Al stresses.

Drought stress is one of the most impactful abiotic stresses to global wheat production. Therefore, identifying key regulators such as the calcineurin B-like protein interacting protein kinase (CIPK) in the signaling cascades known to coordinate developmental cues and environmental stimuli represents a useful approach to improve drought tolerance. However, functional studies have been very limited partly due to the difficulties in prioritizing candidate genes from the large TaCIPK family. To address this issue, we demonstrate a straight-forward strategy by analyzing gene expression patterns in response to phytohormones or stresses and identified TaCIPK19 as a new regulator to improve drought tolerance. The effects of TaCIPK19 on drought tolerance were evaluated in both tobacco and wheat through transgenic approach. Ectopic expression of TaCIPK19 in tobacco greatly improves drought tolerance with enhanced ABA biosynthesis/signaling and ROS scavenging capacity. TaCIPK19 overexpression in wheat also confers the drought tolerance at both seedling and mature stages with enhanced ROS scavenging capacity. Additionally, potential CBL partners interacting with TaCIPK19 were investigated. Collectively, our finding exemplifies a straight-forward approach to facilitate reverse genetics related to abiotic stress improvement and demonstrates TaCIPK19 as a new candidate gene to improve ROS scavenging capacity and drought tolerance, which is useful for genetic improvement and breeding application in wheat.

BACKGROUND: Reverse genetic studies conducted in the plant with a complex or polyploidy genome enriched with large gene families (like wheat) often meet challenges in identifying the key candidate genes related to important traits and prioritizing the genes for functional experiments.
OBJECTIVE: To overcome the above-mentioned challenges of reverse genetics, this work aims to establish an efficient multi-species strategy for genome-wide gene identification and prioritization of the key candidate genes.
METHODS: We established the integrative gene duplication and genome-wide analysis (iGG analysis) as a strategy for pinpointing key candidate genes deserving functional research. The iGG captures the evolution, and the expansion/contraction of large gene families across phylogeny-related species and integrates spatial-temporal expression information for gene function inference. Transgenic approaches were also employed to functional validation.
RESULTS: As a proof-of-concept for the iGG analysis, we took the wheat calcineurin B-like protein-interacting protein kinases (CIPKs) family as an example. We identified CIPKs from seven monocot species, established the orthologous relationship of CIPKs between rice and wheat, and characterized Triticeae-specific CIPK duplicates (e.g., CIPK4 and CIPK17). Integrated with our analysis of CBLs and CBL-CIPK interaction, we revealed that divergent expressions of TaCBLs and TaCIPKs could play an important role in keeping the stoichiometric balance of CBL-CIPK. Furthermore, we validated the function of TaCIPK17-A2 in the regulation of drought tolerance by using transgenic approaches. Overexpression of TaCIPK17 enhanced antioxidant capacity and improved drought tolerance in wheat.
CONCLUSIONS: The iGG analysis leverages evolutionary and comparative genomics of crops with large genomes to rapidly highlight the duplicated genes potentially associated with speciation, domestication and/or particular traits that deserve reverse-genetic functional studies. Through the identification of Triticeae-specific TaCIPK17 duplicates and functional validation, we demonstrated the effectiveness of the iGG analysis and provided a new target gene for improving drought tolerance in wheat.

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.

Plants have acquired sets of highly regulated and complex signaling pathways to respond to unfavorable environmental conditions during evolution. Calcium signaling, as a vital mechanism, enables plants to respond to external stimuli, including abiotic and biotic stresses, and coordinate the basic processes of growth and development. In the present study, two calcium sensor families, CBL and CIPK, were investigated in a halophyte plant, Aeluropus littoralis, with a comprehensive analysis. Here, six AlCBL genes, and twenty AlCIPK genes were studied. The analysis of the gene structure and conserved motifs, as well as physicochemical properties, showed that these genes are highly conserved during evolution. The expression levels of AlCBL genes and AlCIPK genes were evaluated under salt stress in leaf and root tissue. Based on the real-time RT-PCR results, the AlCIPK gene family had a higher variation in mRNA abundance than the AlCBL gene family. AlCIPK genes were found to have a higher abundance in leaves than in roots. The results suggest that the correlation between AlCBL genes and AlCIPK is tissue-specific, and different correlations can be expected in leaves and roots. Based on these correlations, AlCIPK3.1-AlCBL4.1 and AlCIPK1.2-AlCBL4.4 can be co-expressed in the root tissue, while AlCBL10 has the potential to be co-expressed with AlCIPK5, AlCIPK26, and AlCIPK12.3 in the leaf tissue. Our findings reveal valuable information on the structure and function of calcium sensor families in A. littoralis, a halophyte plant, that can be used in future research on the biological function of CBLs and CIPKs on salt stress resistance.

Oryza Sativa is one of the most important food crops in China, which is easily affected by drought during its growth and development. As a member of the calcium signaling pathway, CBL-interacting protein kinase (CIPK) plays an important role in plant growth and development as well as environmental stress. However, there is no report on the function and mechanism of OsCIPK17 in rice drought resistance. We combined transcriptional and metabonomic analysis to clarify the specific mechanism of OsCIPK17 in response to rice drought tolerance. The results showed that OsCIPK17 improved drought resistance of rice by regulating deep roots under drought stress; Response to drought by regulating the energy metabolism pathway and controlling the accumulation of citric acid in the tricarboxylic acid (TCA) cycle; Our exogenous experiments also proved that OsCIPK17 responds to citric acid, and this process involves the auxin metabolism pathway; Exogenous citric acid can improve the drought resistance of overexpression plants. Our research reveals that OsCIPK17 positively regulates rice drought resistance and participates in the accumulation of citric acid in the TCA cycle, providing new insights for rice drought resistance.

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.

CIPKs are a subclass of serine/threonine (Ser/Thr) protein kinases. CBLs are ubiquitous Ca2+ sensors that interact with CIPK with the aid of secondary Ca2+ messengers for regulation of growth and development and response to stresses faced by plants. The divergent roles of the CIPK-CBL interaction in plants include responding to environmental stresses (salt, cold, drought, pH, ABA signaling, and ion toxicity), ion homeostasis (K+, NH4 +, NO3 -, and microelement homeostasis), biotic stress, and plant development. Each member of this gene family produces distinct proteins that help plants adapt to diverse stresses or stimuli by interacting with calcium ion signals. CIPK consists of two structural domains-an N-terminal domain and a C-terminal domain-connected by a junction domain. The N-terminal domain, the site of phosphorylation, is also called the activation domain and kinase domain. The C-terminal, also known as the regulatory domain of CIPK, further comprises NAF/FISL and PPI. CBL comprises four EF domains and conserved PFPF motifs and is the site of binding with the NAF/FISL domain of CIPK to form a CBL-CIPK complex. In addition, we also performed a bibliometric analysis of the CIPK gene family of data extracted from the WoSCC. A total of 95 documents were retrieved, which had been published by 47 sources. The production over time was zigzagged. The top key terms were gene, CIPK, abiotic stress, and gene expression. Beijing Forestry University was the top affiliation, while The Plant Cell was the top source. The genomics and metabolomics of this gene family require more study.

The calcium signaling pathway is critical for plant growth, development, and response to external stimuli. The CBL-CIPK pathway has been well characterized as a calcium-signaling pathway. However, in most reports, only a single function for this module has been described. Here, we examined multiple functions of this module. CIPK showed a similar distribution to that of CBL, and OsCBL and OsCIPK families were retained after experiencing whole genome duplication events through the phylogenetic and synteny analysis. This study found that OsCBL8 negatively regulated rice seed germination and seedling growth by interacting with OsCIPK17 with overexpression and gene editing mutant plants as materials combining plant phenotype, physiological indicators and transcriptome sequencing. This process is likely mediated by OsPP2C77, which is a member of the ABA signaling pathway. In addition, OsCBL mediated the targeting of OsNAC77 and OsJAMYB by OsCIPK17, thus conferring resistance to high temperatures and pathogens in rice. Our work reveals a unique signaling pathway, wherein OsCBL8 interacts with OsCIPK17 and provides rice with multiple resistance while also regulating seedling growth.