Cloning and expression analysis of potassium channel gene NKT3 from Nicotiana tabacum

Potassium (K+) is the predominant inorganic ion of plant cells. K+ channels in higher plant cells play an important role in regulating the influx and efflux of K+ from cells, and activity of these channels might be involved in plant stress resistance. A completely new K+ channel gene of Nicotiana tabacum was obtained through homologous cloning strategy. The complete cDNA sequence was submitted to the National Center for Biotechnology Information (NCBI) GenBank, designated as NKT3 and the accession number is FJ230956. The phylogenetic analysis indicated that NKT3 is located at the branch of weak-inwardly rectifying K+ channels and might be a member of the Shaker family. The spatial and temporal expression of the gene was also investigated. NKT3 is expressed abundantly in the roots, while little in the leaves of N. tabacum. It might be involved in the process of K+ acquirement and release in tobacco roots.Keywords: Potassium channel gene, NKT3, RACE, Nicotiana tabacu

Determination of optimal NH4+/K + concentration and corresponding ratio critical for growth of tobacco seedlings in a hydroponic system

Inherently, ammonium (NH4+) is critical for plant growth; however, its toxicity suppresses potassium (K+) uptake and vice-versa. Hence, attaining a nutritional balance between these two ions (NH4+ and K+) becomes imperative for the growth of tobacco seedlings. Therefore, we conducted a 15-day experimental study on tobacco seedlings exposed to different concentrations (47 treatments) of NH4+/K+ at different corresponding 12 ratios simultaneously in a hydroponic system. Our study aimed at establishing the optimal NH4+-K+ concentration and the corresponding ratio required for optimal growth of different tobacco plant organs during the seedling stage. The controls were the baseline for comparison in this study. Plants with low or excessive NH4+-K+ concentration had leaf chlorosis or dark greenish colouration, stunted whole plant part biomass, and thin roots. We found that adequate K+ supply is a pragmatic way to mitigate NH4+-induced toxicity in tobacco plants. The optimal growth for tobacco leaf and root was attained at NH4+-K+ concentrations 2-2 mM (ratio 1:1), whereas stem growth was optimal at NH4+-K+ 1-2 mM (1:2). The study provided an insight into the right combination of NH4+/K+ that could mitigate or prevent NH4+ or K+ stress in the tobacco seedlings

A pH-stable alkaline pectate lyase produced by the newly identified strain Bacillus altitudinis CAS-WZS-08

Objectives: Thermal and pH stabilities are extremely important for the application of pectate lyase. In this study, we aimed to obtain a strain that is able to produce pectate lyase with good pH stability. Methods: In this study, screening for pectate lyase was performed using plate assays. Fermentation parameters for pectate lyase production were optimized utilizing a single variable optimization. To get insight into the pectate lyase, its enzyme property, purification, identification, and application were performed. Results: Bacillus altitudinis CAS-WZS-08, producing pectate lyase with good pH stability, was isolated. The optimal fermentation conditions of CAS-WZS-08 are 4 g/L pectin, 20 g/L yeast extract, 2% inoculum size, pH 7.0, and 33 °C, which the production of pectate lyase can reach up to 0.71 ± 0.001 U/mL. The optimal pH and temperature of the pectate lyase were 10.0 and 60 °C, respectively. Stored at 4 °C, the pectate lyase was able to keep its full enzyme activity for 24 h under a wide range of pH (4.0–10.0) condition. With pH 10.0, this pectate lyase was stable under 30-45 °C. In addition, it can be activated by Mn2+, Cu2+, Co2+, and Ca2+, while inhibited by Fe3+, Ba2+, and Mg2+. Later, the electrophoretic pure protein was acquired through ammonium sulfate precipitation, cation exchange column, and Sephadex G-75. Liquid chromatography tandem-mass spectrometry (LC/MS-MS) further confirmed that the purified protein was pectate lyase with a molecular weight of ∼ 40 kDa. At last, the result of pectate lyase in extracting apple juice demonstrated that it has an excellent juice extraction ability. Conclusion: This study provides an excellent pH-stable pectate lyase with good thermal stability that is a potential candidate for industrial applications

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

The Arabidopsis Calcium-Dependent Protein Kinases (CDPKs) and Their Roles in Plant Growth Regulation and Abiotic Stress Responses

As a ubiquitous secondary messenger in plant signaling systems, calcium ions (Ca2+) play essential roles in plant growth and development. Within the cellular signaling network, the accurate decoding of diverse Ca2+ signal is a fundamental molecular event. Calcium-dependent protein kinases (CDPKs), identified commonly in plants, are a kind of vital regulatory protein deciphering calcium signals triggered by various developmental and environmental stimuli. This review chiefly introduces Ca2+ distribution in plant cells, the classification of Arabidopsis thaliana CDPKs (AtCDPKs), the identification of the Ca2+-AtCDPK signal transduction mechanism and AtCDPKs’ functions involved in plant growth regulation and abiotic stress responses. The review presents a comprehensive overview of AtCDPKs and may contribute to the research of CDPKs in other plants

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

The Calcium Sensor CBL-CIPK Is Involved in Plant’s Response to Abiotic Stresses

Abiotic stress halts the physiological and developmental process of plant. During stress condition, CBL-CIPK complex is identified as a primary element of calcium sensor to perceive environmental signals. Recent studies established that this complex regulates downstream targets like ion channels and transporters in adverse stages conditions. Crosstalks between the CBL-CIPK complex and different abiotic stresses can extend our research area, which can improve and increase the production of genetically modified crops in response to abiotic stresses. How this complex links with environmental signals and creates adjustable circumstances under unfavorable conditions is now one of the burning issues. Diverse studies are already underway to delineate this signalling mechanism underlying different interactions. Therefore, up to date experimental results should be concisely published, thus paving the way for further research. The present review will concisely recapitulate the recent and ongoing research progress of positive ions (Mg2+, Na+, and K+), negative ions (NO3-, PO4-), and hormonal signalling, which are evolving from accumulating results of analyses of CBL and CIPK loss- or gain-of-function experiments in different species along with some progress and perspectives of our works. In a word, this review will give one step forward direction for more functional studies in this area

Mechanisms and Physiological Roles of the CBL-CIPK Networking System in Arabidopsis thaliana

Calcineurin B-like protein (CBL)-CBL-interacting protein kinase (CIPK) network is one of the vital regulatory mechanisms which decode calcium signals triggered by environmental stresses. Although the complicated regulation mechanisms and some novel functions of CBL-CIPK signaling network in plants need to be further elucidated, numerous advances have been made in its roles involved in the abiotic stresses. This review chiefly introduces the progresses about protein interaction, classification and expression pattern of different CBLs and CIPKs in Arabidopsis thaliana, summarizes the physiological roles of CBL-CIPK pathway while pointing out some new research ideas in the future, and finally presents some unique perspectives for the further study. The review might provide new insights into the functional characterization of CBL-CIPK pathway in Arabidopsis, and contribute to a deeper understanding of CBL-CIPK network in other plants or stresses