CBL-interacting Protein Kinases Research Articles

Target of rapamycin regulates potassium uptake in Arabidopsis and potato

Potassium (K) is an essential inorganic nutrient needed by plants for their growth and development. The conserved target of rapamycin (TOR) kinase, a well-known nutrition signaling integrator, has crucial roles in regulating growth and development in all eukaryotes. Emerging evidence suggests that TOR is a core regulator of nutrient absorption and utilization in plants. However, it is still unclear whether there is a causative link between the TOR pathway and potassium absorption. Here, we show that the expression of some potassium transporters and channels was regulated by TOR, and the suppression of TOR activity significantly affected potassium uptake in Arabidopsis and potato. Furthermore, we discovered that a Type 2A phosphatase-associated protein of 46 kDa (TAP46), a direct TOR downstream effector, could interact with CBL-interacting protein kinase 23 (CIPK23) in Arabidopsis and potato. In Arabidopsis, the K+ channel AKT1 conducting K+ uptake was significantly regulated by Calcineurin B-like Calcium Sensor Protein 1/9 (CBL1/9)-CIPK23 modules. We found that the cbl1cbl9, cipk23 (lks1-2 and lks1-3), and akt1 mutants were more hyposensitive to the TOR inhibitor than the wild-type, and the TOR inhibitor induced the downregulation of K+ uptake rate in the wild-type more than in these mutants. In addition, the overexpression of CIPK23 could effectively restore the defects in growth and potassium uptake induced by the TOR inhibitors. Thus, our work reveals a link between TOR signaling and CIPK23 and provides new insight into the regulation of potassium uptake in plants.

The Ca2+ -regulated protein kinase CIPK1 integrates plant responses to phosphate deficiency in Arabidopsis thaliana.

Phosphate (Pi) deficiency severely restricts plant growth and development, as Pi is an essential macronutrient. Calcium (Ca2+ ) is a ubiquitous second messenger in plants; calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK) are signalling pathways that act as an important Ca2+ signalling network which integrates plants to fine tune the response to stress; however, whether CIPK are involved in Pi deficiency stress remains largely unknown. In this study, we carried out a reverse genetic strategy to screen T-DNA insertion mutants of CIPK isoforms under Pi deficiency in Arabidopsis thaliana. Then Pi content, transcription of phosphate starvation-induced (PSI) genes, acid phosphatase activity and hydrogen peroxide were determined in the wild-type (WT) and cipk1 mutant, respectively. The phenotype of CIPK1 complementation lines was analysed. The cipk1 mutant had a more sensitive phenotype, with lower root elongation and root length, and decreased Pi content compared with the WT under Pi deficiency. Moreover, CIPK1 mutation caused phosphate starvation-induced (PSI) genes to be significantly induced under Pi deficiency. Histological staining demonstrated that the cipk1 mutant had increased acid phosphatase activity and hydrogen peroxide concentration under Pi deficiency. By using the yeast two-hybrid system, we further demonstrated the interaction between CIPK1 and the WRKY transcription factors, WRKY6 and WRKY42. Overall, we demonstrate that CIPK1 is involved in the Pi deficiency signalling pathway in A. thaliana, revealing the important role of Ca2+ in the Pi nutrition signalling pathway, and potentially providing a theoretical foundation for molecular breeding of crops with better Pi utilization efficiency.

Isolation and characterization of Salt Overly Sensitive family genes in spinach

The Salt Overly Sensitive (SOS) pathway regulates intracellular sodium ion homeostasis as a salt-stress response in plants. This pathway involves three main genes designated as SOS1, SOS2 and SOS3, which are members of the Na+ /H+ exchanger (NHX), CBL-interacting protein kinase (CIPK) and Calcineurin B-like (CBL) gene families, respectively. To identify and characterize SOS genes in spinach (Spinacia oleracea), a species of the Amaranthaceae family, we conducted genome-wide identification and phylogenetic analyses of NHX, CIPK and CBL genes from four Amaranthaceae species, Arabidopsis and rice. Most Amaranthaceae genes exhibited orthologous relationships with Arabidopsis and/or rice, except a clade of Vac-type Amaranthaceae NHX genes. Phylogenetic analyses also revealed gene gain/loss events in Amaranthaceae species and the intron-less to intron-rich evolution of CIPK genes. A bacterial protein-rooted CIPK tree allowed naming most of the phylogenetic clades based on their evolutionary history. Single S. oleracea (So) SOS1, SOS2 and SOS3 proteins were identified. Direct protein-protein interaction was observed between SoSOS2 and SoSOS3 but not between SoSOS2 and SoSOS1 based on yeast two-hybrid assay. This may suggest distinct modes of action of spinach SOS proteins compared to Arabidopsis SOS proteins. Unlike SoSOS1 and SoSOS2, which were expressed at similar or higher levels in leaves than roots, SoSOS3 expression was significantly higher in roots than leaves, suggesting its greater importance in roots. The expression of SoSOS3 was upregulated in both roots and leaves under salinity compared to the control; however, SoSOS1 was only upregulated in roots. Thus, this study demonstrated the conservation of SOS pathway genes in spinach and also highlighted the complexity of SOS signaling in Amaranthaceae species.

Characterization of CBL–CIPK signaling complexes and their involvement in cold response in tea plant

Calcineurin B-like (CBL) proteins, a class of Ca2+-binding proteins, play vital roles in calcium signal transduction by interacting specifically with CBL-interacting protein kinases (CIPKs), and these two gene families and their interacting complexes are involved in regulating plant responses to various environmental stimuli. In the present study, eight CBL and 25 CIPK genes were identified in tea plant and divided into four and five subfamilies, respectively. Analysis of the expression of these genes in response to abiotic stresses (mature leaves treated with cold, salinity, and PEG and young shoots treated with cold) revealed that CsCBL1/3/5 and CsCIPK1/4/5/6a/7/8/10b/10c/12/14a/19/23a/24 could be induced by at least two stresses. Under cold stress, CsCBL9 and CsCIPK4/6a/6b/7/11/14b/19/20 were upregulated in both mature leaves and young shoots, CsCBL1/3/5 and CsCIPK1/8/10a/10b/10c/12/14a/23a/24 were induced only in mature leaves, and CsCIPK5/25 were induced only in young shoots. Yeast two-hybrid analysis showed that CsCBL1 could interact with CsCIPK1/10b/12 but not with CsCIPK6a/7/11/14b/20. CsCBL9 was found to interact with CsCIPK1/10b/12/14b but not with CsCIPK6a/7/11/20. These results suggest divergent responses to cold stress regulated by CBL–CIPK complexes between tea plant and Arabidopsis, as well as between mature leaves and young shoots in tea plant. A model of Ca2+–CsCBL–CsCIPK module-mediated abiotic stress signaling in tea plant is proposed.

Nitrate Signaling, Functions, and Regulation of Root System Architecture: Insights from Arabidopsis thaliana.

Root system architecture (RSA) is required for the acquisition of water and mineral nutrients from the soil. One of the essential nutrients, nitrate (NO3−), is sensed and transported by nitrate transporters NRT1.1 and NRT2.1 in the plants. Nitrate transporter 1.1 (NRT1.1) is a dual-affinity nitrate transporter phosphorylated at the T101 residue by calcineurin B-like interacting protein kinase (CIPKs); it also regulates the expression of other key nitrate assimilatory genes. The differential phosphorylation (phosphorylation and dephosphorylation) strategies and underlying Ca2+ signaling mechanism of NRT1.1 stimulate lateral root growth by activating the auxin transport activity and Ca2+-ANR1 signaling at the plasma membrane and the endosomes, respectively. NO3− additionally functions as a signal molecule that forms a signaling system, which consists of a vast array of transcription factors that control root system architecture that either stimulate or inhibit lateral and primary root development in response to localized and high nitrate (NO3−), respectively. This review elucidates the so-far identified nitrate transporters, nitrate sensing, signal transduction, and the key roles of nitrate transporters and its downstream transcriptional regulatory network in the primary and lateral root development in Arabidopsis thaliana under stress conditions.

NH4+ Toxicity, Which Is Mainly Determined by the High NH4+/K+ Ratio, Is Alleviated by CIPK23 in Arabidopsis.

Ammonium (NH4+) toxicity is always accompanied by ion imbalances, and NH4+ and potassium (K+) exhibit a competitive correlation in their uptake and transport processes. In Arabidopsis thaliana, the typical leaf chlorosis phenotype in the knockout mutant of calcineurin B-like interacting protein kinase 23 (CIPK23) is high-NH4+-dependent under low-K+ condition. However, the correlation of K+ and NH4+ in the occurrence of leaf chlorosis in the cipk23 mutant has not been deeply elucidated. Here, a modified hydroponic experimental system with different gradients of NH4+ and K+ was applied. Comparative treatments showed that NH4+ toxicity, which is triggered mainly by the high ratio of NH4+ to K+ (NH4+/K+ ≥ 10:1 for cipk23) but not by the absolute concentrations of the ions, results in leaf chlorosis. Under high NH4+/K+ ratios, CIPK23 is upregulated abundantly in leaves and roots, which efficiently reduces the leaf chlorosis by regulating the contents of NH4+ and K+ in plant shoots, while promoting the elongation of primary and lateral roots. Physiological data were obtained to further confirm the role CIPK23 in alleviating NH4+ toxicity. Taken all together, CIPK23 might function in different tissues to reduce stress-induced NH4+ toxicity associated with high NH4+/K+ ratios by regulating the NH4+–K+ balance in Arabidopsis.

A calcium signalling network activates vacuolar K+ remobilization to enable plant adaptation to low-K environments.

Potassium (K) is an essential nutrient, but levels of the free K ions (K+) in soil are often limiting, imposing a constant stress on plants. We have discovered a calcium (Ca2+)-dependent signalling network, consisting of two calcineurin B-like (CBL) Ca2+ sensors and a quartet of CBL-interacting protein kinases (CIPKs), which plays a key role in plant response to K+ starvation. The mutant plants lacking two CBLs (CBL2 and CBL3) were severely stunted under low-K conditions. Interestingly, the cbl2 cbl3 mutant was normal in K+ uptake but impaired in K+ remobilization from vacuoles. Four CIPKs-CIPK3, 9, 23 and 26-were identified as partners of CBL2 and CBL3 that together regulate K+ homeostasis through activating vacuolar K+ efflux to the cytoplasm. The vacuolar two-pore K+ (TPK) channels were directly activated by the vacuolar CBL-CIPK modules in a Ca2+-dependent manner, presenting a mechanism for the activation of vacuolar K+ remobilization that plays an important role in plant adaptation to K+ deficiency.

The CBL-CIPK Calcium Signaling Network: Unified Paradigm from 20 Years of Discoveries.

Calcium (Ca2+) serves as an essential nutrient as well as a signaling agent in all eukaryotes. 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). Interactions between CBLs and CIPKs constitute a signaling network that enables information integration and physiological coordination in response to a variety of extracellular cues such as nutrient deprivation and abiotic stresses. Studies in the past two decades have established a unified paradigm that illustrates the functions of CBL-CIPK complexes in controlling membrane transport through targeting transporters and channels in the plasma membrane and tonoplast.

The Calcium Sensor CBL2 and Its Interacting Kinase CIPK6 Are Involved in Plant Sugar Homeostasis via Interacting with Tonoplast Sugar Transporter TST2.

Calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK)-mediated calcium signaling has been widely reported to function in plant development and various stress responses, particularly in ion homeostasis. Sugars are the most important primary metabolites, and thus sugar homeostasis requires precise regulation. Here, we describe a CBL2-CIPK6-Tonoplast-Localized Sugar Transporter2 (TST2) molecular module in cotton (Gossypium hirsutum) that regulates plant sugar homeostasis, in particular Glc homeostasis. GhCIPK6 is recruited to the tonoplast by GhCBL2 and interacts with the tonoplast-localized sugar transporter GhTST2. Overexpression of either GhCBL2, GhCIPK6, or GhTST2 was sufficient to promote sugar accumulation in transgenic cotton, whereas RNAi-mediated knockdown of GhCIPK6 expression or CRISPR-Cas9-mediated knockout of GhTST2 resulted in significantly decreased Glc content. Moreover, mutation of GhCBL2 or GhTST2 in GhCIPK6-overexpressing cotton reinstated sugar contents comparable to wild-type plants. Heterologous expression of GhCIPK6 in Arabidopsis (Arabidopsis thaliana) also promoted Glc accumulation, whereas mutation of AtTST1/2 in GhCIPK6-overexpressing Arabidopsis similarly reinstated wild-type sugar contents, thus indicating conservation of CBL2-CIPK6-TST2-mediated sugar homeostasis among different plant species. Our characterization of the molecular players behind plant sugar homeostasis may be exploited to improve sugar contents and abiotic stress resistance in plants.

Evolutionary strategies drive a balance of the interacting gene products for the CBL and CIPK gene families.

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.

Recognition and Activation of the Plant AKT1 Potassium Channel by the Kinase CIPK23.

Plant growth largely depends on the maintenance of adequate intracellular levels of potassium (K+). The families of 10 Calcineurin B-Like (CBL) calcium sensors and 26 CBL-Interacting Protein Kinases (CIPKs) of Arabidopsis (Arabidopsis thaliana) decode the calcium signals elicited by environmental inputs to regulate different ion channels and transporters involved in the control of K+ fluxes by phosphorylation-dependent and -independent events. However, the detailed molecular mechanisms governing target specificity require investigation. Here, we show that the physical interaction between CIPK23 and the noncanonical ankyrin domain in the cytosolic side of the inward-rectifier K+ channel AKT1 regulates kinase docking and channel activation. Point mutations on this domain specifically alter binding to CIPK23, enhancing or impairing the ability of CIPK23 to regulate channel activity. Our data demonstrate the relevance of this protein-protein interaction that contributes to the formation of a complex between CIPK23/CBL1 and AKT1 in the membrane for the proper regulation of K+ transport.

Genome-Wide Characterization and Analysis of CIPK Gene Family in Two Cultivated Allopolyploid Cotton Species: Sequence Variation, Association with Seed Oil Content, and the Role of GhCIPK6.

Calcineurin B-like protein-interacting protein kinases (CIPKs), as key regulators, play an important role in plant growth and development and the response to various stresses. In the present study, we identified 80 and 78 CIPK genes in the Gossypium hirsutum and G. barbadense, respectively. The phylogenetic and gene structure analysis divided the cotton CIPK genes into five groups which were classified into an exon-rich clade and an exon-poor clade. A synteny analysis showed that segmental duplication contributed to the expansion of Gossypium CIPK gene family, and purifying selection played a major role in the evolution of the gene family in cotton. Analyses of expression profiles showed that GhCIPK genes had temporal and spatial specificity and could be induced by various abiotic stresses. Fourteen GhCIPK genes were found to contain 17 non-synonymous single nucleotide polymorphisms (SNPs) and co-localized with oil or protein content quantitative trait loci (QTLs). Additionally, five SNPs from four GhCIPKs were found to be significantly associated with oil content in one of the three field tests. Although most GhCIPK genes were not associated with natural variations in cotton oil content, the overexpression of the GhCIPK6 gene reduced the oil content and increased C18:1 and C18:1+C18:1d6 in transgenic cotton as compared to wild-type plants. In addition, we predicted the potential molecular regulatory mechanisms of the GhCIPK genes. In brief, these results enhance our understanding of the roles of CIPK genes in oil synthesis and stress responses.

Transcriptome Analysis of Rice Roots in Response to Root-Knot Nematode Infection.

Meloidogyne incognita and Meloidogyne graminicola are root-knot nematodes (RKNs) infecting rice (Oryza sativa L.) roots and severely decreasing yield, whose mechanisms of action remain unclear. We investigated RKN invasion and development in rice roots through RNA-seq transcriptome analysis. The results showed that 952 and 647 genes were differently expressed after 6 (invasion stage) and 18 (development stage) days post inoculation, respectively. Gene annotation showed that the differentially expressed genes were classified into diverse metabolic and stress response categories. Furthermore, phytohormone, transcription factor, redox signaling, and defense response pathways were enriched upon RKN infection. RNA-seq validation using qRT-PCR confirmed that CBL-interacting protein kinase (CIPK) genes (CIPK5, 8, 9, 11, 14, 23, 24, and 31) as well as brassinosteroid (BR)-related genes (OsBAK1, OsBRI1, D2, and D11) were altered by RKN infection. Analysis of the CIPK9 mutant and overexpressor indicated that the RKN populations were smaller in cipk9 and larger in CIPK9 OX, while more galls were produced in CIPK9 OX plant roots than the in wild-type roots. Significantly fewer numbers of second-stage infective juveniles (J2s) were observed in the plants expressing the BR biosynthesis gene D2 mutant and the BR receptor BRI1 activation-tagged mutant (bri1-D), and fewer galls were observed in bri1-D roots than in wild-type roots. The roots of plants expressing the regulator of ethylene signaling ERS1 (ethylene response sensor 1) mutant contained higher numbers of J2s and developed more galls compared with wild-type roots, suggesting that these signals function in RKN invasion or development. Our findings broaden our understanding of rice responses to RKN invasion and provide useful information for further research on RKN defense mechanisms.

Role of an Amur grape CBL-interacting protein kinase VaCIPK02 in drought tolerance by modulating ABA signaling and ROS production

Calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK) play critical roles in mediating plant responses to various biotic and abiotic stresses. However, the underlying molecular mechanisms by which these complex functions and the potential cross talk between the diverse downstream signaling networks are regulated still remain elusive. Here we characterized a VaCIPK02 from Vitis amurensis, and show it functions in salt and drought stresses. We have identified that, at least, the domain at C terminus of VaCIPK02 involved in transcriptional activation. VaCIPK02 could strongly interact with four Ca2+ sensor proteins, VaCBL1, 4, 5 and 8, respectively. A Y2H-based screen to isolate genes encoding VaCIPK02-interacting proteins identified 12 additional partners, including an ABA receptor PYL9, which interacts with VaCIPK02 in vivo. Constitutive expression of VaCIPK02 in Arabidopsis showed extremely sensitive to salt stress, but displayed increased tolerance to drought stress accompanied by ABA-hypersensitive stomata closure, improved water-conserving capacity, and increased cellular ABA levels. Moreover, overexpression of VaCIPK02 caused reduced cellular levels of ABA-induced reactive oxygen species (ROS) by regulating the expression levels of many ABA-responsive genes to scavenge and modulate ROS levels. Our results provide evidence for the involvement of VaCIPK02 along with its diverse interacting proteins in regulating salt and drought stress signal pathway.

Role of the Arabidopsis calcineurin B-like protein-interacting protein kinase CIPK21 in plant cold stress tolerance

The Arabidopsis calcineurin B-like protein-interacting protein kinase CIPK21 gene (AtCIPK21) plays important roles in cell metabolism, plant development, and abiotic stress responses. However, the function of the AtCIPK21 gene in cold stress tolerance in plant cells is not fully understood. In this study, we use cell cultures of three plant species including rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and white pine (Pinus strobus L.), as well as Agrobacterium tumefaciens strain GV3101 harboring pBI-AtCIPK21 to generate transgenic cell lines. After confirmation of integration of the AtCIPK21 gene into the genome by polymerase chain reaction (PCR), southern blotting, and northern blotting analyses, the rice, cotton, and pine AtCIPK21 transgenic cell lines were used to examine cold stress tolerance. The experimental results demonstrated that overexpression of the AtCIPK21 gene enhanced cold stress tolerance of transgenic cells by increasing cell viability and cell growth rate, decreasing lipid peroxidation and ion leakage, increasing the content of polyamines, as well as elevating the activity of antioxidative enzymes. In rice cells, AtCIPK21 increases expression of Ca2+-dependent protein kinase (CPK) genes and mitogen-activated protein kinase (MAPK) genes under cold stress. These results indicated that overexpression of the AtCIPK21 gene in plant cells improved cold stress tolerance by elevating polyamines content, antioxidative enzyme activity, and expression of CPK genes and MAPK genes. Overexpression of the AtCIPK21 gene may be a valuable approach for engineering plant cold stress tolerance.

A CBL-interacting protein kinase AdCIPK5 confers salt and osmotic stress tolerance in transgenic tobacco

CBL interacting protein kinases play important roles in adaptation to stress conditions. In the present study, we isolated a CBL-interacting protein kinase homolog (AdCIPK5) from a wild peanut (Arachis diogoi) with similarity to AtCIPK5 of Arabidopsis. Expression analyses in leaves of the wild peanut showed AdCIPK5 induction by exogenous signaling molecules including salicylic acid, abscisic acid and ethylene or abiotic stress factors like salt, PEG and sorbitol. The recombinant AdCIPK5-GFP protein was found to be localized to the nucleus, plasma membrane and cytoplasm. We overexpressed AdCIPK5 in tobacco plants and checked their level of tolerance to biotic and abiotic stresses. While wild type and transgenic plants displayed no significant differences to the treatment with the phytopathogen, Phytophthora parasitica pv nicotianae, the expression of AdCIPK5 increased salt and osmotic tolerance in transgenic plants. Analysis of different physiological parameters revealed that the transgenic plants maintained higher chlorophyll content and catalase activity with lower levels of H2O2 and MDA content during the abiotic stress conditions. AdCIPK5 overexpression also contributed to the maintenance of a higher the K+/Na+ ratio under salt stress. The enhanced tolerance of transgenic plants was associated with elevated expression of stress-related marker genes; NtERD10C, NtERD10D, NtNCED1, NtSus1, NtCAT and NtSOS1. Taken together, these results indicate that AdCIPK5 is a positive regulator of salt and osmotic stress tolerance.

Screening and Identification of NsylCBL Family Members Interacting with Protein Kinase NsylCIPK24a in Nicotiana Sylvestris

The CIPK (CBL-interacting protein kinase) kinase family in plants is a type of serine/threonine protein kinase family. The members of this family interact with the upstream CBL protein (Calcineurin B-like protein) to form the CBL-CIPK signal system, involved in regulating plant growth and development as well as stress response processes. Early research found that AtCIPK24 interacts with AtCBL4 and AtCBL10, respectively, to activate downstream target proteins to respond to high salt stress. Earlier in this study, NsylCIPK24a homologous to AtCIPK24 from Arabidopsis thaliana was obtained from Nicotiana sylvestris , but CBL family members interacting with NsylCIPK24a and its specific function are unclear. Therefore, a genome-wide prediction of CBL family members of N. sylvestris was carried out; Predicted NsylCBL genes were cloned by RT-PCR, and the analyses of gene structure, protein conserved domain and expression pattern were then conducted. The NsylCBL members interacting with NsylCIPK24a were screened by the yeast two-hybrid system. Results showed that there are 12 potential NsylCBL genes in N. sylvestris, and all of them were cloned successfully. Four members including NsylCBL4, NsylCBL5, NsylCBL9 and NsylCBL10 could interact with NsylCIPK24a in yeast. There might be some similar CBL-CIPK pathways in tobacco plants, compared with Arabidopsis. The study provides experimental data for the functional identification of NsylCIPK24a , and increases understanding of CBL-CIPK network in N. sylvestris.

Identification and Functional Analysis of Tomato CIPK Gene Family.

The calcineurin B-like interacting protein kinase (CIPK) protein family is a critical protein family in plant signaling pathways mediated by Ca2+, playing a pivotal role in plant stress response and growth. However, to the best of our knowledge, no study of the tomato CIPK gene family in response to abiotic stress has been reported. In this study, 22 members of the tomato CIPK gene family were successfully identified by using a combination of bioinformatics techniques and molecular analyses. The expression level of each member of tomato CIPK gene family under abiotic stress (low temperature, high salt, drought treatment) was determined by qRT-PCR. Results indicated that tomato CIPK demonstrated different degrees of responding to various abiotic stresses, and changes in SlCIPK1 and SlCIPK8 expression level were relatively apparent. The results of qRT-PCR showed that expression levels of SlCIPK1 increased significantly in early stages of cold stress, and the expression level of SlCIPK8 increased significantly during the three treatments at different time points, implicating Solanum lycopersicum CIPK1(SlCIPK1) and Solanum lycopersicum CIPK8 (SlCIPK8) involvement in abiotic stress response. SlCIPK1 and SlCIPK8 were silenced using Virus-induced gene silencing (VIGS), and physiological indexes were detected by low temperature, drought, and high salt treatment. The results showed that plants silenced by SlCIPK1 and SlCIPK8 at the later stage of cold stress were significantly less resistant to cold than wild-type plants. SlCIPK1 and SlCIPK8 silenced plants had poor drought resistance, indicating a relationship between SlCIPK1 and SlCIPK8 with response to low temperature and drought resistance. This is the first study to uncover the nucleotide sequence for tomato CIPK family members and systematically study the changes of tomato CIPK family members under abiotic stress. Here, we investigate the CIPK family’s response under abiotic stress providing understanding into the signal transduction pathway. This study provides a theoretical basis for elucidating the function of tomato CIPK at low temperature and its molecular mechanism of regulating low temperatures.

Characterization and tissue-specific as well as heat-stress expression analysis ofCBL-interacting protein kinase genes in Dimocarpuslongan Lour

The CBL-interacting protein kinase (CIPK) gene family plays important roles in plant growth, development, and responses to abiotic stresses. To date, the CIPKs have been extensively identified and characterized in various plants. However, there have been few studies on CIPKs in Dimocarpus longan Lour (D. longan). In this study we identified eight putative CIPKs in D. longan (DlCIPKs) according to RNA-seq data. The physicochemical properties, subcellular localization, conserved motifs, phylogenetic relationships, glycosylation and phosphorylation sites, and gene ontology annotation were analyzed using bioinformatics tools. The tissue-specific and heat-stress-responsive expressions were systematically investigated as well. Ten motifs widely maintained by DlCIPKs were identified. All eight DlCIPKs identified from D. longan and the 50 CIPKs selected from Zea mays, Oryza sativa, Sorghum bicolor, Vitis vinifera, Populus trichocarpa, and Arabidopsis thaliana were categorized together into seven groups; group 7 was the largest and contained 20 CIPKs. Localizations of these eight DlCIPKs were predicted in the plasma membrane, cytoplasm, chloroplast thylakoid membrane, or nucleus and were predicted to differ. All eight DlCIPKs were predicted to be phosphorylated but with different numbers of phosphorylation sites. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that among the eight DlCIPKs, only DlCIPK1, DlCIPK5, and DlCIPK8 were differently expressed; notably, between root and leaf tissues. All eight DlCIPKs responded to heat treatments. The expressions of DlCIPK1, DlCIPK4, DlCIPK5, and DlCIPK6 in D. longan leaves were upregulated to the maximum level after 1 h of heat treatment. In contrast, the expression levels of DlCIPK2, DlCIPK3, DlCIPK7, and DlCIPK8 increased significantly after 4 h of heat treatment. The results of this study will enrich our knowledge of DlCIPKs and establish a foundation for enhancing tolerance to abiotic stresses through genetic engineering in D. longan.

GABA-Alleviated Oxidative Injury Induced by Salinity, Osmotic Stress and their Combination by Regulating Cellular and Molecular Signals in Rice.

This study was conducted in order to determine the effect of priming with γ-aminobutyric acid (GABA) at 0.5 mM on rice (Oryza sativa L.) seed germination under osmotic stress (OS) induced by polyethylene glycol (30 g/L PEG 6000); and salinity stress (S, 150 mM NaCl) and their combination (OS+S). Priming with GABA significantly alleviated the detrimental effects of OS, S and OS+S on seed germination and seedling growth. The photosynthetic system and water relation parameters were improved by GABA under stress. Priming treatment significantly increased the GABA content, sugars, protein, starch and glutathione reductase. GABA priming significantly reduced Na+ concentrations, proline, free radical and malonaldehyde and also significantly increased K+ concentration under the stress condition. Additionally, the activities of antioxidant enzymes, phenolic metabolism-related enzymes, detoxification-related enzymes and their transcription levels were improved by GABA priming under stress. In the GABA primed-plants, salinity stress alone resulted in an obvious increase in the expression level of Calcineurin B-like Protein-interacting protein Kinases (CIPKs) genes such as OsCIPK01, OsCIPK03, OsCIPK08 and OsCIPK15, and osmotic stress alone resulted in obvious increase in the expression of OsCIPK02, OsCIPK07 and OsCIPK09; and OS+S resulted in a significant up-regulation of OsCIPK12 and OsCIPK17. The results showed that salinity, osmotic stresses and their combination induced changes in cell ultra-morphology and cell cycle progression resulting in prolonged cell cycle development duration and inhibitory effects on rice seedlings growth. Hence, our findings suggested that the high tolerance to OS+S is closely associated with the capability of GABA priming to control the reactive oxygen species (ROS) level by inducing antioxidant enzymes, secondary metabolism and their transcription level. This knowledge provides new evidence for better understanding molecular mechanisms of GABA-regulating salinity and osmotic-combined stress tolerance during rice seed germination and development.