The CIPK23 protein kinase represses SLAC1-type anion channels in Arabidopsis guard cells and stimulates stomatal opening.

Guard cells control the opening of stomatal pores in the leaf surface, with the use of a network of protein kinases and phosphatases. Loss of function of the CBL-interacting protein kinase 23 (CIPK23) was previously shown to decrease the stomatal conductance, but the molecular mechanisms underlying this response still need to be clarified. CIPK23 was specifically expressed in Arabidopsis guard cells, using an estrogen-inducible system. Stomatal movements were linked to changes in ion channel activity, determined with double-barreled intracellular electrodes in guard cells and with the two-electrode voltage clamp technique in Xenopus oocytes. Expression of the phosphomimetic variant CIPK23T190D enhanced stomatal opening, while the natural CIPK23 and a kinase-inactive CIPK23K60N variant did not affect stomatal movements. Overexpression of CIPK23T190D repressed the activity of S-type anion channels, while their steady-state activity was unchanged by CIPK23 and CIPK23K60N . We suggest that CIPK23 enhances the stomatal conductance at favorable growth conditions, via the regulation of several ion transport proteins in guard cells. The inhibition of SLAC1-type anion channels is an important facet of this response.

Finding Balance in Adversity: Nitrate Signaling as the Key to Plant Growth, Resilience, and Stress Response.

To effectively adapt to changing environments, plants must maintain a delicate balance between growth and resistance or tolerance to various stresses. Nitrate, a significant inorganic nitrogen source in soils, not only acts as an essential nutrient but also functions as a critical signaling molecule that regulates multiple aspects of plant growth and development. In recent years, substantial advancements have been made in understanding nitrate sensing, calcium-dependent nitrate signal transmission, and nitrate-induced transcriptional cascades. Mounting evidence suggests that the primary response to nitrate is influenced by environmental conditions, while nitrate availability plays a pivotal role in stress tolerance responses. Therefore, this review aims to provide an overview of the transcriptional and post-transcriptional regulation of key components in the nitrate signaling pathway, namely, NRT1.1, NLP7, and CIPK23, under abiotic stresses. Additionally, we discuss the specificity of nitrate sensing and signaling as well as the involvement of epigenetic regulators. A comprehensive understanding of the integration between nitrate signaling transduction and abiotic stress responses is crucial for developing future crops with enhanced nitrogen-use efficiency and heightened resilience.

Sequence Characteristics and Expression Analysis of GhCIPK23 Gene in Upland Cotton (Gossypium hirsutum L.).

CIPK (calcineurin B-like-interacting protein kinase) is a kind of serine/threonine protein kinase widely existing in plants, and it plays an important role in plant growth and development and stress response. To better understand the biological functions of the GhCIPK23 gene in upland cotton, the coding sequence (CDS) of the GhCIPK23 gene was cloned in upland cotton, and its protein sequence, evolutionary relationship, subcellular localization, expression pattern and cis-acting elements in the promoter region were analyzed. Our results showed that the full-length CDS of GhCIPK23 was 1368 bp, encoding a protein with 455 amino acids. The molecular weight and isoelectric point of this protein were 50.83 KDa and 8.94, respectively. The GhCIPK23 protein contained a conserved N-terminal protein kinase domain and C-terminal regulatory domain of the CIPK gene family member. Phylogenetic tree analysis demonstrated that GhCIPK23 had a close relationship with AtCIPK23, followed by OsCIPK23, and belonged to Group A with AtCIPK23 and OsCIPK23. The subcellular localization experiment indicated that GhCIPK23 was located in the plasma membrane. Tissue expression analysis showed that GhCIPK23 had the highest expression in petals, followed by sepals, and the lowest in fibers. Stress expression analysis showed that the expression of the GhCIPK23 gene was in response to drought, salt, low-temperature and exogenous abscisic acid (ABA) treatment, and had different expression patterns under different stress conditions. Further cis-acting elements analysis showed that the GhCIPK23 promoter region had cis-acting elements in response to abiotic stress, phytohormones and light. These results established a foundation for understanding the function of GhCIPK23 and breeding varieties with high-stress tolerance in cotton.

Regulation of Root Nutrient Transporters by CIPK23: 'One Kinase to Rule Them All'.

Protein kinases constitute essential regulatory components in the majority of cellular processes in eukaryotic cells. The CBL-INTERACTING PROTEIN KINASE (CIPK) family of plant protein kinases functions in calcium (Ca2+)-related signaling pathways and is therefore involved in the response to a wide variety of signals in plants. By covalently linking phosphate groups to their target proteins, CIPKs regulate the activity of downstream targets, their localization, their stability and their ability to interact with other proteins. In Arabidopsis, the CIPK23 kinase has emerged as a major hub driving root responses to diverse environmental stresses, including drought, salinity and nutrient imbalances, such as potassium, nitrate and iron deficiencies, as well as ammonium, magnesium and non-iron metal toxicities. This review will chiefly report on the prominent roles of CIPK23 in the regulation of plant nutrient transporters and on the underlying molecular mechanisms. We will also discuss the different scenarios explaining how a single promiscuous kinase, such as CIPK23, may convey specific responses to a myriad of signals.

STOP1 Regulates LKS1 Transcription and Coordinates K+/NH4+ Balance in Arabidopsis Response to Low-K+ Stress.

Potassium and nitrogen are essential mineral elements for plant growth and development. The protein kinase LKS1/CIPK23 is involved in both K+ and NH4+ uptake in Arabidopsis root. The transcripts of LKS1 can be induced by low K+ (0.1 mM) and high NH4+ (30 mM); however, the molecular mechanism is still unknown. In this study, we isolated the transcription factor STOP1 that positively regulates LKS1 transcription in Arabidopsis responses to both low-K+ and high-NH4+ stresses. STOP1 proteins can directly bind to the LKS1 promoter, promoting its transcription. The stop1 mutants displayed a leaf chlorosis phenotype similar to lks1 mutant when grown on low-K+ and high-NH4+ medium. On the other hand, STOP1 overexpressing plants exhibited a similar tolerant phenotype to LKS1 overexpressing plants. The transcript level of STOP1 was only upregulated by low K+ rather than high NH4+; however, the accumulation of STOP1 protein in the nucleus was required for the upregulation of LKS1 transcripts in both low-K+ and high-NH4+ responses. Our data demonstrate that STOP1 positively regulates LKS1 transcription under low-K+ and high-NH4+ conditions; therefore, LKS1 promotes K+ uptake and inhibits NH4+ uptake. The STOP1/LKS1 pathway plays crucial roles in K+ and NH4+ homeostasis, which coordinates potassium and nitrogen balance in plants in response to external fluctuating nutrient levels.

Sensitive to Proton Rhizotoxicity1 Regulates Salt and Drought Tolerance of Arabidopsis thaliana through Transcriptional Regulation of CIPK23.

The transcription factor sensitive to proton rhizotoxicity 1 (STOP1) regulates multiple stress tolerances. In this study, we confirmed its involvement in NaCl and drought tolerance. The root growth of the T-DNA insertion mutant of STOP1 (stop1) was sensitive to NaCl-containing solidified MS media. Transcriptome analysis of stop1 under NaCl stress revealed that STOP1 regulates several genes related to salt tolerance, including CIPK23. Among all available homozygous T-DNA insertion mutants of the genes suppressed in stop1, only cipk23 showed a NaCl-sensitive root growth phenotype comparable to stop1. The CIPK23 promoter had a functional STOP1-binding site, suggesting a strong CIPK23 suppression led to NaCl sensitivity of stop1. This possibility was supported by in planta complementation of CIPK23 in the stop1 background, which rescued the short root phenotype under NaCl. Both stop1 and cipk23 exhibited a drought tolerant phenotype and increased abscisic acid-regulated stomatal closure, while the complementation of CIPK23 in stop1 reversed these traits. Our findings uncover additional pleiotropic roles of STOP1 mediated by CIPK23, which regulates various ion transporters including those regulating K+-homeostasis, which may induce a trade-off between drought tolerance and other traits.

Phosphorylation by CIPK23 regulates the high-affinity Mn transporter NRAMP1 in Arabidopsis.

Manganese (Mn) is essential for plants but is toxic when taken up in excess. To maintain Mn homeostasis, the root Mn transporter natural resistance associated macrophage protein 1 (NRAMP1) cycles from the plasma membrane to endosomes upon phosphorylation. To identify the kinase involved, a split-luciferase screening was carried out between NRAMP1 and kinases of the CIPK family and identified CIPK23 as a partner of NRAMP1. The interaction was confirmed by split-mCitrine bimolecular fluorescence complementation and co-immunoprecipitation assays. In vitro phosphorylation assays pinpointed two CIPK23 target residues in NRAMP1, among which serine 20, important for endocytosis. Interestingly, Mn-induced internalization of NRAMP1 was unaffected by cipk23 mutation suggesting a potential redundancy between CIPK23 and other kinase(s). How CIPK23 could regulate NRAMP1 in response to Mn availability is discussed.

A transcription factor STOP1-centered pathway coordinates ammonium and phosphate acquisition in Arabidopsis.

Phosphorus (P) is an indispensable macronutrient required for plant growth and development. Natural phosphate (Pi) reserves are finite, and a better understanding of Pi utilization by crops is therefore vital for worldwide food security. Ammonium has long been known to enhance Pi acquisition efficiency in agriculture; however, the molecular mechanisms coordinating Pi nutrition and ammonium remains unclear. Here, we reveal that ammonium is a novel initiator that stimulates the accumulation of a key regulatory protein, STOP1, in the nuclei of Arabidopsis root cells under Pi deficiency. We show that Pi deficiency promotes ammonium uptake mediated by AMT1 transporters and causes rapid acidification of the root surface. Rhizosphere acidification-triggered STOP1 accumulation activates the excretion of organic acids, which help to solubilize Pi from insoluble iron or calcium phosphates. Ammonium uptake by AMT1 transporters is downregulated by a CIPK23 protein kinase whose expression is directly modulated by STOP1 when ammonium reaches toxic levels. Taken together, we have identified a STOP1-centered regulatory network that links external ammonium with efficient Pi acquisition from insoluble phosphate sources. These findings provide a framework for developing possible strategies to improve crop production by enhancing the utilization of non-bioavailable nutrients in soil.

The CBL1/9-CIPK23-AKT1 complex is essential for low potassium response in cassava.

Cassava is a food crop and an important energy crop worldwide. However, its yield and quality are easily affected by low K+ stress, and the molecular mechanism of potassium channel is unknown in cassava. Herein, we revealed that calcineurin B-like 1/9 (MeCBL1/9)-CBL-interacting protein kinase 23 (MeCIPK23)-K+ TRANSPORTER1 (MeAKT1) complex plays an important role in low potassium response in cassava. Firstly, this study verified the in vivo role of MeAKT1 in K+ uptake in yeast. Secondly, we found that MeCBL1, MeCBL9, MeCIPK23 and MeAKT1 are involved in the absorption of K+ in cassava, and MeCBL1/9-CIPK23 complex is essential for MeAKT1-mediated K+ uptake. Moreover, MeCBL1/9-MeCIPK23-MeAKT1 showed different expression in different cassava varieties contrasting in the resistance to low K+ stress. Taken together, this study provides new insights into further improvement of K+ uptake in cassava.

The CBL-Interacting Protein Kinase NtCIPK23 Positively Regulates Seed Germination and Early Seedling Development in Tobacco (Nicotiana tabacum L.).

CBL-interacting protein kinase (CIPK) family is a unique group of serine/threonine protein kinase family identified in plants. Among this family, AtCIPK23 and its homologs in some plants are taken as a notable group for their importance in ions transport and stress responses. However, there are limited reports on their roles in seedling growth and development, especially in Solanaceae plants. In this study, NtCIPK23, a homolog of AtCIPK23 was cloned from Nicotiana tabacum. Expression analysis showed that NtCIPK23 is mainly expressed in the radicle, hypocotyl, and cotyledons of young tobacco seedlings. The transcriptional level of NtCIPK23 changes rapidly and spatiotemporally during seed germination and early seedling growth. To study the biological function of NtCIPK23 at these stages, the overexpressing and CRISPR/Cas9-mediated knock-out (ntcipk23) tobacco lines were generated. Phenotype analysis indicated that knock-out of NtCIPK23 significantly delays seed germination and the appearance of green cotyledon of young tobacco seedling. Overexpression of NtCIPK23 promotes cotyledon expansion and hypocotyl elongation of young tobacco seedlings. The expression of NtCIPK23 in hypocotyl is strongly upregulated by darkness and inhibited under light, suggesting that a regulatory mechanism of light might underlie. Consistently, a more obvious difference in hypocotyl length among different tobacco materials was observed in the dark, compared to that under the light, indicating that the upregulation of NtCIPK23 contributes greatly to the hypocotyl elongation. Taken together, NtCIPK23 not only enhances tobacco seed germination, but also accelerate early seedling growth by promoting cotyledon greening rate, cotyledon expansion and hypocotyl elongation of young tobacco seedlings.

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.

FIP1 Plays an Important Role in Nitrate Signaling and Regulates CIPK8 and CIPK23 Expression in Arabidopsis.

Unraveling the molecular mechanisms of nitrate regulation and deciphering the underlying genetic network is vital for elucidating nitrate uptake and utilization in plants. Such knowledge could lead to the improvement of nitrogen-use efficiency in agriculture. Here, we report that the FIP1 gene (factor interacting with poly(A) polymerase 1) plays an important role in nitrate signaling in Arabidopsis thaliana. FIP1 encodes a putative core component of the polyadenylation factor complex. We found that FIP1 interacts with the cleavage and polyadenylation specificity factor 30-L (CPSF30-L), which is also an essential player in nitrate signaling. The induction of nitrate-responsive genes following nitrate treatment was inhibited in the fip1 mutant. The nitrate content was also reduced in fip1 seedlings due to their decreased nitrate uptake activity. Furthermore, the nitrate content was higher in the roots but lower in the roots of fip1, which may result from the downregulation of NRT1.8 and the upregulation of the nitrate assimilation genes. In addition, qPCR analyses revealed that FIP1 negatively regulated the expression of CIPK8 and CIPK23, two protein kinases involved in nitrate signaling. In the fip1 mutant, the increased expression of CIPK23 may affect nitrate uptake, resulting in its lower nitrate content. Genetic and molecular evidence suggests that FIP1 and CPSF30-L function in the same nitrate-signaling pathway, with FIP1 mediating signaling through its interaction with CPSF30-L and its regulation of CIPK8 and CIPK23. Analysis of the 3'-UTR of NRT1.1 showed that the pattern of polyadenylation sites was altered in the fip1 mutant. These findings add a novel component to the nitrate regulation network and enhance our understanding of the underlying mechanisms for nitrate signaling.