Regulation of ion homeostasis under salt stress

When under salt stress, plants maintain a high concentration of K(+) and a low concentration of Na(+) in the cytosol. They do this by regulating the expression and activity of K(+) and Na(+) transporters and of H(+) pumps that generate the driving force for transport. Although salt-stress sensors remain elusive, some of the intermediary signaling components have been identified. Evidence suggests that a protein kinase complex consisting of the myristoylated calcium-binding protein SOS3 and the serine/threonine protein kinase SOS2 is activated by a salt-stress-elicited calcium signal. The protein kinase complex then phosphorylates and activates various ion transporters, such as the plasma membrane Na(+)/H(+) antiporter SOS1.     

Ethylene and nitric oxide are involved in maintaining ion homeostasis in <Emphasis Type="Italic">Arabidopsis</Emphasis> callus under salt stress

In the present study, the role of ethylene in nitric oxide (NO)-mediated protection by modulating ion homeostasis in Arabidopsis callus under salt stress was investigated. Results showed that the ethylene-insensitive mutant etr1-3 was more sensitive to salt stress than the wild type (WT). Under 100 mM NaCl, etr1-3 callus displayed a greater electrolyte leakage and Na⁺/K⁺ ratio but a lower plasma membrane (PM) H⁺-ATPase activity compared to WT callus. Application of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) or sodium nitroprusside (SNP, a NO donor) alleviated NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and an increased PM H⁺-ATPase activity in WT callus but not in etr1-3 callus. The SNP actions in NaCl stress were attenuated by a specific NO scavenger or an ethylene biosynthesis inhibitor in WT callus. Under 100 mM NaCl, the NO accumulation and ethylene emission appeared at early time, and NO production greatly stimulated ethylene emission in WT callus. In addition, ethylene induced the expression of PM H⁺-ATPase genes under salt stress. The recovery experiment showed that NaCl-induced injury was reversible, as signaled by the similar recovery of Na⁺/K⁺ ratio and PM H⁺-ATPase activity in WT callus. Taken together, the results indicate that ethylene and NO cooperate in stimulating PM H⁺-ATPase activity to modulate ion homeostasis for salt tolerance, and ethylene may be a part of the downstream signal molecular in NO action.     

Zinc homeostasis is involved in unfolded protein response under salt stress

Accumulation of unfolded protein or misfolded protein causes endoplasmic reticulum (ER) stress. Increased salt concentration activates a stress response pathway in the ER in Arabidopsis thaliana to induce the expression of several salt stress response genes, leading to a more optimal protein folding environment in the ER. In addition, some salt stress-regulated proteins require zinc for their activity, including some zinc-dependent DNA binding proteins and zinc-finger proteins. In a recent study, we reported that ZTP29, a putative zinc transporter at the ER membrane, is involved in the response to salt stress through regulation of zinc level in the ER to induce the UPR pathway. In this addendum, we propose a testable hypothesis for the role of ZTP29 in the response to salt stress via the regulation of zinc levels in the ER.Key words: zinc, ER stress, unfolded protein response, salt stress, arabidopsisHigh salinity is a common abiotic stress that adversely affects plant growth and crop production.¹ Plants must sense the stress and transduce stress signals to activate response pathways leading to adaptation to, or tolerance of, the abiotic stress in salt environment.² Salt stress activates a stress response pathway in the endoplasmic reticulum (ER) in Arabidopsis thaliana, indicating that the adaptation of plants to salt stress involves ER stress signal regulation.^3,4 There is limited understanding of molecular mechanisms on ER stress in plants, as compared to yeast and mammalian cells. bZIP60, bZIP28, bZIP17 are three membrane-associated basic domain/leucine zipper (bZIP) factors, which have been reported as candidates for ER-folding proteins in plants.^5–7 BiP acts as a general chaperone in the ER lumen, due to its ability to discriminate between properly folded and unfolded protein structures.⁸ Unfolded or misfolded proteins are retained in the ER and form stable complexes with BiP and other ER resident chaperones.⁹ Zinc deficiency induces unfolded protein response (UPR) in most eukaryotes.¹⁰ Zinc is an important trace element, which participates in physiological and biochemical process in vivo. The requirement of zinc for proper ER function is evolutionarily conserved.     

A barley mutant with improved salt tolerance through ion homeostasis and ROS scavenging under salt stress

To investigate key regulatory components and genes with great impact on salt tolerance, near isogenic or mutant lines with distinct salinity tolerance are suitable genetic materials to simplify and dissect the complex genes networks. In this study, we evaluated responses of a barley mutant genotype (73-M4-30), in comparison with its wild-type background (Zarjou) under salt stress. Although the root growth of both genotypes was significantly decreased by exposure to sodium chloride (NaCl), the effect was greater in the wild type. The chlorophyll content decreased under salt stress for the wild type, but no change occurred in the mutant. The mutant maintained the steady-state level of [K⁺] and significantly lower [Na⁺] concentrations in roots and higher [K⁺]/[Na⁺] ratio in shoots under salt conditions. The catalase (CAT), peroxidase (POD) activity, and proline content were higher in the mutant than those in the wild type under controlled conditions. The soluble proline was higher after 24 h of salt stress in roots of the mutant but was higher after 96 h of salt stress in the wild type. The CAT and POD activity of the mutant increased under salt stress which was as a coincidence to lower levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents. The ratio of dry-to-fresh weight of the roots increased for the mutant under salt stress which was as a result of the higher phenylalanine ammonia-lyase (PAL) gene expression and peroxidase activity and involved in cell wall lignification. Consequently, it seems that ion homeostasis and increased peroxidase activity have led to salt tolerance in the mutant’s genotype.     

盐胁迫下植物细胞离子稳态重建机制

土壤盐渍化是困扰世界粮食产量的一大难题。在盐胁迫环境中,植物获得耐盐能力的一个重要策略是建立新的离子稳态(ionic homeostasis)。盐胁迫下植物细胞离子稳态依赖于膜转运蛋白(泵、载体和离子通道)。利用蛋白质的生化功能分析和突变体功能互补等方法,目前已克隆和鉴定了许多参与离子稳态重建的膜转运蛋白。综述了盐胁迫下植物细胞离子稳态重建的最新研究进展。     

An enhancer mutant of Arabidopsis salt overly sensitive 3 mediates both ion homeostasis and the oxidative stress response

The myristoylated calcium sensor SOS3 and its interacting protein kinase, SOS2, play critical regulatory roles in salt tolerance. Mutations in either of these proteins render Arabidopsis thaliana plants hypersensitive to salt stress. We report here the isolation and characterization of a mutant called enh1-1 that enhances the salt sensitivity of sos3-1 and also causes increased salt sensitivity by itself. ENH1 encodes a chloroplast-localized protein with a PDZ domain at the N-terminal region and a rubredoxin domain in the C-terminal part. Rubredoxins are known to be involved in the reduction of superoxide in some anaerobic bacteria. The enh1-1 mutation causes enhanced accumulation of reactive oxygen species (ROS), particularly under salt stress. ROS also accumulate to higher levels in sos2-1 but not in sos3-1 mutants. The enh1-1 mutation does not enhance sos2-1 phenotypes. Also, enh1-1 and sos2-1 mutants, but not sos3-1 mutants, show increased sensitivity to oxidative stress. These results indicate that ENH1 functions in the detoxification of reactive oxygen species resulting from salt stress by participating in a new salt tolerance pathway that may involve SOS2 but not SOS3.     

外源cGMP调控盐胁迫下黑麦草种子萌发机制

c GMP(cyclic guanosine-3',5'-monophosphate)是一类环化核苷酸,能响应植物非生物胁迫的信号分子。以c GMP的膜透性类似物8-Br-cGMP为供体,研究c GMP对盐胁迫下黑麦草种子萌发及生理指标的影响。通过计算萌发指标、测定种子萌发过程中各项生理指标,探讨c GMP对黑麦草种子萌发时耐盐机制的作用。结果表明:低浓度(50 mmol/L)盐胁迫能促进黑麦草种子的萌发,随着盐浓度(100、150、200、250 mmol/L)的增加黑麦草种子的萌发率逐渐降低甚至抑制其萌发。而加入20μmol/L c GMP能缓解100 mmol/L Na Cl胁迫对黑麦草种子造成的伤害,使黑麦草种子的发芽率、发芽势、发芽指数、活力指数分别提高了7.4%、133.3%、52.1%、104.2%。此外,研究发现加入20μmol/Lc GMP能够促进100 mmol/L Na Cl盐胁迫下黑麦草萌发期根的生长,比单独100 mmol/L Na Cl盐胁迫处理下的黑麦草植株全长、根长、叶长分别提高了2.5、2.8倍和2.6倍,种子在萌发过程中可溶性糖、可溶性蛋白含量、淀粉酶活性,脯氨酸含量分别提高了47.9%、15.7%、94.3%、117.4%,而淀粉含量、MDA含量、电导率,O_2~-产生速率分别降低了40.9%、128.7%、88.6%、211.9%。说明20μmol/Lc GMP通过提高可溶性糖,可溶性蛋白含量及脯氨酸含量,同时减少MDA、O_2~-的产生,以此缓解盐胁迫对黑麦草种子的伤害、同时促进淀粉水解,从而加速种子萌发。     

The calcium sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis

Calcium serves as a critical messenger in many adaptation and developmental processes. Cellular calcium signals are detected and transmitted by sensor molecules such as calcium-binding proteins. In plants, the calcineurin B-like protein (CBL) family represents a unique group of calcium sensors and plays a key role in decoding calcium transients by specifically interacting with and regulating a family of protein kinases (CIPKs). We report here that the CBL protein CBL10 functions as a crucial regulator of salt tolerance in Arabidopsis. Cbl10 mutant plants exhibited significant growth defects and showed hypersensitive cell death in leaf tissues under high-salt conditions. Interestingly, the Na(+) content of the cbl10 mutant, unlike other salt-sensitive mutants identified thus far, was significantly lower than in the wild type under either normal or high-salt conditions, suggesting that CBL10 mediates a novel Ca(2+)-signaling pathway for salt tolerance. Indeed, the CBL10 protein physically interacts with the salt-tolerance factor CIPK24 (SOS2), and the CBL10-CIPK24 (SOS2) complex is associated with the vacuolar compartments that are responsible for salt storage and detoxification in plant cells. These findings suggest that CBL10 and CIPK24 (SOS2) constitute a novel salt-tolerance pathway that regulates the sequestration/compartmentalization of Na(+) in plant cells. Because CIPK24 (SOS2) also interacts with CBL4 (SOS3) and regulates salt export across the plasma membrane, our study identifies CIPK24 (SOS2) as a multi-functional protein kinase that regulates different aspects of salt tolerance by interacting with distinct CBL calcium sensors.     

Na+-K+ transport in roots under salt stress

Salinity causes billion dollar losses in annual crop production. So far, the main avenue in breeding crops for salt tolerance has been to reduce Na⁺ uptake and transport from roots to shoots. Recently we have demonstrated that retention of cytosolic K⁺ could be considered as another key factor in conferring salt tolerance in plants. A subsequent study has shown that Na⁺-induced K⁺ efflux in barley root epidermis occurs primarily via outward rectifying K⁺ channels (KORC). Surprisingly, expression of KORC was similar in salt- tolerant and sensitive genotypes. However, the former were able to better oppose Na⁺-induced depolarization via enhanced activity of plasma membrane H⁺-ATPase (thus minimizing K⁺ leak from the cytosol). In addition to highly K⁺-selective KORC channels, activities of several types of non-selective cation channels were detected at depolarizing potentials. Here we show that the expression of one of them, NORC, was significantly lower in salt-tolerant genotypes. As NORC is capable of mediating K⁺ efflux coupled to Na⁺ influx, we suggest that the restriction of its activity could be beneficial for plants under salt stress.Key words: salinity tolerance, barley, ion flux, K⁺ homeostasis, KOR, non-selective channels, patch-clamp     

Endoplasmic reticulum stress and alteration in calcium homeostasis are involved in cadmium-induced apoptosis

Cadmium, a toxic environmental contaminant, exerts adverse effects on different cellular pathways such as cell proliferation, DNA damage and apoptosis. In particular, the modulation of Ca(2+) homeostasis seems to have an important role during Cd(2+) injury, but the precise assessment of Ca(2+) signalling still remains poorly understood. We used aequorin-based probes specifically directed to intracellular organelles to study Ca(2+) changes during cadmium injury. We observed that cadmium decreased agonist-evoked endoplasmic reticulum (ER) Ca(2+) signals and caused a 40% inhibition of sarcoplasmic-ER calcium ATPases activity. Moreover, time course experiments correlate morphological alterations, processing of xbp-1 mRNA and caspase-12 activation during cadmium administration. Finally, the time response of ER to cadmium injury was compared with that of mitochondria. In conclusion, we highlighted a novel pathway of cadmium-induced cell death triggered by ER stress and involving caspase-12. Mitochondria and ER pathways seemed to share common time courses and a parallel activation of caspase-12 and caspase-9 seemed likely to be involved in acute cadmium toxicity.     

Fluorescence recovery after photobleaching reveals high cycling dynamics of plasma membrane aquaporins in Arabidopsis roots under salt stress

The constitutive cycling of plant plasma membrane (PM) proteins is an essential component of their function and regulation under resting or stress conditions. Transgenic Arabidopsis plants that express GFP fusions with AtPIP1;2 and AtPIP2;1, two prototypic PM aquaporins, were used to develop a fluorescence recovery after photobleaching (FRAP) approach. This technique was used to discriminate between PM and endosomal pools of the aquaporin constructs, and to estimate their cycling between intracellular compartments and the cell surface. The membrane trafficking inhibitors tyrphostin A23, naphthalene-1-acetic acid and brefeldin A blocked the latter process. By contrast, a salt treatment (100 mm NaCl for 30 min) markedly enhanced the cycling of the aquaporin constructs and modified their pharmacological inhibition profile. Two distinct models for PM aquaporin cycling in resting or salt-stressed root cells are discussed.     

Phosphorylation dynamics of membrane proteins from Arabidopsis roots submitted to salt stress

An excess of NaCl in the soil is detrimental for plant growth. It interferes with mineral nutrition and water uptake and leads to accumulation of toxic ions in the plant. Understanding the response of roots to NaCl stress may facilitate the development of crops with increased tolerance to this and other stresses. Since controls achieved at the posttranslational level are of critical importance for regulating protein function, the present work used a robust label‐free quantitative proteomic methodology to quantify phosphorylation events that affect root membrane proteins in Arabidopsis, in response to short‐term (up to 2 h) NaCl treatments. This work identified 302 proteotypic phosphopeptides including 77 novel phosphorylated sites. NaCl treatment significantly altered the abundance of 74 phosphopeptides, giving novel insights into the regulation of major classes of membrane proteins, including ATPases, sodium transporters, and aquaporins. The data provide a unique access to phosphorylation reprogramming of ionic equilibrium in plant cells under NaCl stress. The use of predictive bioinformatic tools for kinase motifs suggested that root membrane proteins are substrates of cAMP‐dependent protein kinase, cGMP‐dependent protein kinase, and protein kinase C families, also called AGC kinases, arguing for an important role of lipid signaling in abiotic stress responses. It also pointed to cross‐talks between protein kinase families during NaCl stress.     

Upstream kinases of plant SnRKs are involved in salt stress tolerance

Sucrose non‐fermenting 1‐related protein kinases (SnRKs) are important for plant growth and stress responses. This family has three clades: SnRK1, SnRK2 and SnRK3. Although plant SnRKs are thought to be activated by upstream kinases, the overall mechanism remains obscure. Geminivirus Rep‐Interacting Kinase (GRIK)1 and GRIK2 phosphorylate SnRK1s, which are involved in sugar/energy sensing, and the grik1‐1 grik2‐1 double mutant shows growth retardation under regular growth conditions. In this study, we established another Arabidopsis mutant line harbouring a different allele of gene GRIK1 (grik1‐2 grik2‐1) that grows similarly to the wild‐type, enabling us to evaluate the function of GRIKs under stress conditions. In the grik1‐2 grik2‐1 double mutant, phosphorylation of SnRK1.1 was reduced, but not eliminated, suggesting that the grik1‐2 mutation is a weak allele. In addition to high sensitivity to glucose, the grik1‐2 grik2‐1 mutant was sensitive to high salt, indicating that GRIKs are also involved in salinity signalling pathways. Salt Overly Sensitive (SOS)2, a member of the SnRK3 subfamily, is a critical mediator of the response to salinity. GRIK1 phosphorylated SOS2 in vitro, resulting in elevated kinase activity of SOS2. The salt tolerance of sos2 was restored to normal levels by wild‐type SOS2, but not by a mutated form of SOS2 lacking the T168 residue phosphorylated by GRIK1. Activation of SOS2 by GRIK1 was also demonstrated in a reconstituted system in yeast. Our results indicate that GRIKs phosphorylate and activate SnRK1 and other members of the SnRK3 family, and that they play important roles in multiple signalling pathways in vivo.     

Multilevel interactions between ethylene and auxin in Arabidopsis roots

Hormones play a central role in the coordination of internal developmental processes with environmental signals. Herein, a combination of physiological, genetic, cellular, and whole-genome expression profiling approaches has been employed to investigate the mechanisms of interaction between two key plant hormones: ethylene and auxin. Quantification of the morphological effects of ethylene and auxin in a variety of mutant backgrounds indicates that auxin biosynthesis, transport, signaling, and response are required for the ethylene-induced growth inhibition in roots but not in hypocotyls of dark-grown seedlings. Analysis of the activation of early auxin and ethylene responses at the cellular level, as well as of global changes in gene expression in the wild type versus auxin and ethylene mutants, suggests a simple mechanistic model for the interaction between these two hormones in roots, according to which ethylene and auxin can reciprocally regulate each other's biosyntheses, influence each other's response pathways, and/or act independently on the same target genes. This model not only implies existence of several levels of interaction but also provides a likely explanation for the strong ethylene response defects observed in auxin mutants.     

UBX domain-containing proteins are involved in lipid homeostasis and stress responses in Pichia pastoris

Ubiquitin regulatory X (UBX) domain-containing proteins constitute a family of proteins and are substrate adaptors of AAA ATPase Cdc48. UBX proteins can bind to the N-terminal region of Cdc48 to perform endoplasmic reticulum associated protein degradation (ERAD). In this study, we identified two UBX domain-containing proteins, Ubx1 and Ubx2, in Pichia pastoris and found that the two proteins could recover the growth defect of Saccharomyces cerevisiae in ubx2Δ. Our results revealed that Ubx1 and Ubx2 play critical roles in synthesis of unsaturated fatty acids by affecting Spt23. In addition, the results demonstrated that both Ubx1 and Ubx2 are involved in lipid droplet formation and protein degradation. Deletion of UBX1 led to increased sensitivity to oxidative stress and disruption of UBX2 impaired cell viability under osmotic stress. The phenotypes of ubx1Δ+UBX2, ubx2Δ+UBX1 and ubx1Δubx2Δ and RNA-seq data suggested that Ubx1 and Ubx2 play different roles in cell functions, and the roles of Ubx1 may be more numerous than Ubx2. In summary, our findings provide new insights into the relationship between lipid homeostasis and cell functions in the oil-producing organism P. pastoris.