Differential regulation of two Arabidopsis type III phosphatidylinositol 4-kinase isoforms. A regulatory role for the pleckstrin homology domain

Here, we compare the regulation and localization of the Arabidopsis type III phosphatidylinositol (PtdIns) 4-kinases, AtPI4Kalpha1 and AtPI4Kbeta1, in Spodoptera frugiperda (Sf9) insect cells. We also explore the role of the pleckstrin homology (PH) domain in regulating AtPI4Kalpha1. Recombinant kinase activity was found to be differentially sensitive to PtdIns-4-phosphate (PtdIns4P), the product of the reaction. The specific activity of AtPI4Kalpha1 was inhibited 70% by 0.5 mm PtdIns4P. The effect of PtdIns4P was not simply due to charge because AtPI4Kalpha1 activity was stimulated approximately 50% by equal concentrations of the other negatively charged lipids, PtdIns3P, phosphatidic acid, and phosphatidyl-serine. Furthermore, inhibition of AtPI4Kalpha1 by PtdIns4P could be alleviated by adding recombinant AtPI4Kalpha1 PH domain, which selectively binds to PtdIns4P (Stevenson et al., 1998). In contrast, the specific activity of AtPI4Kbeta1, which does not have a PH domain, was stimulated 2-fold by PtdIns4P but not other negatively charged lipids. Visualization of green fluorescent protein fusion proteins in insect cells revealed that AtPI4Kalpha1 was associated primarily with membranes in the perinuclear region, whereas AtPI4Kbeta1 was in the cytosol and associated with small vesicles throughout the cytoplasm. Expression of AtPI4Kalpha1 without the PH domain in the insect cells compromised PtdIns 4-kinase activity and caused mislocalization of the kinase. The green fluorescent protein-PH domain alone was associated with intracellular membranes and the plasma membrane. In vitro, the PH domain appeared to be necessary for association of AtPI4Kalpha1 with fine actin filaments. These studies support the idea that the Arabidopsis type III PtdIns 4-kinases are responsible for distinct phosphoinositide pools.     

The involvement of wheat U‐box E3 ubiquitin ligase TaPUB1 in salt stress tolerance

U‐box E3 ubiquitin ligases play important roles in the ubiquitin/26S proteasome machinery and in abiotic stress responses. TaPUB1‐overexpressing wheat (Triticum aestivum L.) were generated to evaluate its function in salt tolerance. These plants had more salt stress tolerance during seedling and flowering stages, whereas the TaPUB1‐RNA interference (RNAi)‐mediated knock‐down transgenic wheat showed more salt stress sensitivity than the wild type (WT). TaPUB1 overexpression upregulated the expression of genes related to ion channels and increased the net root Na⁺ efflux, but decreased the net K⁺ efflux and H⁺ influx, thereby maintaining a low cytosolic Na⁺/K⁺ ratio, compared with the WT. However, RNAi‐mediated knock‐down plants showed the opposite response to salt stress. TaPUB1 could induce the expression of some genes that improved the antioxidant capacity of plants under salt stress. TaPUB1 also interacted with TaMP (Triticum aestivum α‐mannosidase protein), a regulator playing an important role in salt response in yeast and in plants. Thus, low cytosolic Na⁺/K⁺ ratios and better antioxidant enzyme activities could be maintained in wheat with overexpression of TaPUB1 under salt stress. Therefore, we conclude that the U‐box E3 ubiquitin ligase TaPUB1 positively regulates salt stress tolerance in wheat.     

The Arabidopsis UDP‐glycosyltransferases UGT79B2 and UGT79B3, contribute to cold,salt and drought stress tolerance via modulating anthocyanin accumulation

The plant family 1 UDP‐glycosyltransferases (UGTs) are the biggest GT family in plants, which are responsible for transferring sugar moieties onto a variety of small molecules, and control many metabolic processes; however, their physiological significance in planta is largely unknown. Here, we revealed that two Arabidopsis glycosyltransferase genes, UGT79B2 and UGT79B3, could be strongly induced by various abiotic stresses, including cold, salt and drought stresses. Overexpression of UGT79B2/B3 significantly enhanced plant tolerance to low temperatures as well as drought and salt stresses, whereas the ugt79b2/b3 double mutants generated by RNAi (RNA interference) and CRISPR‐Cas9 strategies were more susceptible to adverse conditions. Interestingly, the expression of UGT79B2 and UGT79B3 is directly controlled by CBF1 (CRT/DRE‐binding factor 1, also named DREB1B) in response to low temperatures. Furthermore, we identified the enzyme activities of UGT79B2/B3 in adding UDP‐rhamnose to cyanidin and cyanidin 3‐O‐glucoside. Ectopic expression of UGT79B2/B3 significantly increased the anthocyanin accumulation, and enhanced the antioxidant activity in coping with abiotic stresses, whereas the ugt79b2/b3 double mutants showed reduced anthocyanin levels. When overexpressing UGT79B2/B3 in tt18 (transparent testa 18), a mutant that cannot synthesize anthocyanins, both genes fail to improve plant adaptation to stress. Taken together, we demonstrate that UGT79B2 and UGT79B3, identified as anthocyanin rhamnosyltransferases, are regulated by CBF1 and confer abiotic stress tolerance via modulating anthocyanin accumulation.     

Rational Design of Novel Phosphoinositide 3‐Kinase Gamma (PI3Kγ) Selective Inhibitors: A Computational Investigation Integrating 3D‐QSAR,Molecular Docking and Molecular Dynamics Simulation

Phosphoinositide 3‐kinase gamma (PI3Kγ) draws an increasing attention due to its link with deadly cancer, chronic inflammation and allergy. But the development of PI3Kγ selective inhibitors is still a challenging endeavor because of the high sequence homology with the other PI3K isoforms. In order to acquire valuable information about the interaction mechanism between potent inhibitors and PI3Kγ, a series of PI3Kγ isoform‐selective inhibitors were analyzed by a systematic computational method, combining 3D‐QSAR, molecular docking, molecular dynamic (MD) simulations, free energy calculations and decomposition. The general structure–activity relationships were revealed and some key residues relating to selectivity and high activity were highlighted. It provides precious guidance for rational virtual screening, modification and design of selective PI3Kγ inhibitors. Finally, ten novel inhibitors were optimized and P10 showed satisfactory predicted bioactivity, demonstrating the feasibility to develop potent PI3Kγ inhibitors through this computational modeling and optimization.     

HbCIPK2, a novel CBL-interacting protein kinase from halophyte Hordeum brevisubulatum, confers salt and osmotic stress tolerance

Protein kinases play an important role in regulating the response to abiotic stress in plant. CIPKs are plant‐specific signal transducers, and some members have been identified. However, the precise functions of novel CIPKs still remain unknown. Here we report that HbCIPK2 is a positive regulator of salt and osmotic stress tolerance. HbCIPK2 was screened out of the differentially expressed fragments from halophyte Hordeum brevisubulatum by cDNA‐AFLP technique, and was a single‐copy gene without intron. Expression of HbCIPK2 was increased by salt, drought and ABA treatment. HbCIPK2 is mainly localized to the plasma membrane and nucleus. Ectopic expression of 35S:HbCIPK2 not only rescued the salt hypersensitivity in Arabidopsis mutant sos2‐1, but also enhanced salt tolerance in Arabidopsis wild type, and exhibited tolerance to osmotic stress during germination. The HbCIPK2 contributed to the ability to prevent K⁺ loss in root and to accumulate less Na⁺ in shoot resulting in K⁺/Na⁺ homeostasis and protection of root cell from death, which is consistent with the gene expression profile of HbCIPK2‐overexpressing lines. These findings imply possible novel HbCIPK2‐mediated salt signalling pathways or networks in H. brevisubulatum.     

Ectopic expression of wheat TaCIPK14, encoding a calcineurin B‐like protein‐interacting protein kinase,confers salinity and cold tolerance in tobacco

Calcineurin B‐like protein‐interacting protein kinases (CIPKs) are components of Ca²⁺ signaling in responses to abiotic stresses. In this work, the full‐length cDNA of a novel CIPK gene (TaCIPK14) was isolated from wheat and was found to have significant sequence similarity to OsCIPK14/15. Subcellular localization assay revealed the presence of TaCIPK14 throughout the cell. qRT‐PCR analysis showed that TaCIPK14 was upregulated under cold conditions or when treated with salt, PEG or exogenous stresses related signaling molecules including ABA, ethylene and H₂O₂. Transgenic tobaccos overexpressing TaCIPK14 exhibited higher contents of chlorophyll and sugar, higher catalase activity, while decreased amounts of H₂O₂ and malondialdehyde, and lesser ion leakage under cold and salt stresses. In addition, overexpression also increased seed germination rate, root elongation and decreased Na⁺ content in the transgenic lines under salt stress. Higher expression of stress‐related genes was observed in lines overexpressing TaCIPK14 compared to controls under stress conditions. In summary, these results suggested that TaCIPK14 is an abiotic stress‐responsive gene in plants.     

Overexpression of <Emphasis Type="Italic">CsNMAPK</Emphasis> in tobacco enhanced seed germination under salt and osmotic stresses

In this research, biological function of CsNMAPK, encoding a mitogen-activated protein kinase of cucumber, was investigated under salt and osmotic stresses. Northern blot analysis showed that the expression of CsNMAPK was induced by salt and osmotic stresses in the cucumber root. In order to determine whether CsNMAPK was involved in plant tolerance to salt and osmotic stresses, transgenic tobacco plants constitutively overexpressing CsNMAPK were generated. Northern and Western blot analysis showed that strong signals were detected in the RNA and protein samples extracted from transgenic lines, whereas no signal was detected in the wild type tobacco, indicating that CsNMAPK was successfully transferred into tobacco genome and overexpressed. The results of seed germination showed that germination rates of transgenic lines were significantly higher than wild type under high salt and osmotic stresses. In addition, seed growth of transgenic lines was much better than wild type under salt and osmotic stresses. These results indicated that overexpression of CsNMAPK positively regulated plant tolerance to salt and osmotic stresses.     

Arabidopsis C3HC4‐RING finger E3 ubiquitin ligase AtAIRP4 positively regulates stress‐responsive abscisic acid signaling

Degradation of proteins via the ubiquitin system is an important step in many stress signaling pathways in plants. E3 ligases recognize ligand proteins and dictate the high specificity of protein degradation, and thus, play a pivotal role in ubiquitination. Here, we identified a gene, named Arabidopsis thaliana abscisic acid (ABA)‐insensitive RING protein 4 (AtAIRP4), which is induced by ABA and other stress treatments. AtAIRP4 encodes a cellular protein with a C3HC4‐RING finger domain in its C‐terminal side, which has in vitro E3 ligase activity. Loss of AtAIRP4 leads to a decrease in sensitivity of root elongation and stomatal closure to ABA, whereas overexpression of this gene in the T‐DNA insertion mutant atairp4 effectively recovered the ABA‐associated phenotypes. AtAIRP4 overexpression plants were hypersensitive to salt and osmotic stresses during seed germination, and showed drought avoidance compared with the wild‐type and atairp4 mutant plants. In addition, the expression levels of ABA‐ and drought‐induced marker genes in AtAIRP4 overexpression plants were markedly higher than those in the wild‐type and atairp4 mutant plants. Hence, these results indicate that AtAIRP4 may act as a positive regulator of ABA‐mediated drought avoidance and a negative regulator of salt tolerance in Arabidopsis.     

Arranged marriage in lipid signalling? The limited choices of PtdIns(4,5)P2 in finding the right partner

Inositol‐containing phospholipids (phosphoinositides, PIs) control numerous cellular processes in eukaryotic cells. For plants, a key involvement of PIs has been demonstrated in the regulation of membrane trafficking, cytoskeletal dynamics and in processes mediating the adaptation to changing environmental conditions. Phosphatidylinositol‐4,5‐bisphosphate (PtdIns(4,5)P₂) mediates its cellular functions via binding to various alternative target proteins. Such downstream targets of PtdIns(4,5)P₂ are characterised by the possession of specific lipid‐binding domains, and binding of the PtdIns(4,5)P₂ ligand exerts effects on their activity or localisation. The large number of potential alternative binding partners – and associated cellular processes – raises the question how alternative or even contrapuntal effects of PtdIns(4,5)P₂ are orchestrated to enable cellular function. This article aims to provide an overview of recent insights and new views on how distinct functional pools of PtdIns(4,5)P₂ are generated and maintained. The emerging picture suggests that PtdIns(4,5)P₂ species containing different fatty acids influence the lateral mobility of the lipids in the membrane, possibly enabling specific interactions of PtdIns(4,5)P₂ pools with certain downstream targets. PtdIns(4,5)P₂ pools with certain functions might also be defined by protein–protein interactions of PI4P 5‐kinases, which pass PtdIns(4,5)P₂ only to certain downstream partners. Individually or in combination, PtdIns(4,5)P₂ species and specific protein–protein interactions of PI4P 5‐kinases might contribute to the channelling of PtdIns(4,5)P₂ signals towards specific functional effects. The dynamic nature of PI‐dependent signalling complexes with specific functions is an added challenge for future studies of plant PI signalling.