Salt Stress Signaling Research Articles

Tomato Inducer of CBF Expression 1 (SlICE1) is Involved in Cold and Salt Stress Signaling

Aims: Inducer of CBF Expression 1 (ICE1), which is one of basic Helix-Loop-Helix type transcription factors, has important roles in regulation of cold stress-indiced genes of plants. Sample: To investigate functions of tomato ICE1 in cold and salt tolerance, c index (SI), immunochemical assay of endogenous ICE1 protein and RT-PCR of coldinducible genes were conducted with tomato plants. Methodology: Tomato plants grown for 4 weeks were subjected to cold (4oC) and salt (0.2 M NaCl) in the presence or the absence of cell signaling inhibitors. An antibody was raised against ICE1 specific epitoipe. Immunoblot with the anti-ICE1 antibody was carried out with extractions of tomato plants treated by cold and salt stresses. The expression profiles of tomato ICE1 (SlICE1) and other cold-inducible genes including LeCBF1/2/3 and SlTPS1 were analyzed by semi-quantitative RT-PCR. Results: An ICE1-related protein with molecular masses of approximately 55 is induced in tomato plant under chilling and salt stresses. The expression of a tomato ICE1 gene (SlICE1) under chilling stress was maintained at a constant level in contrast to the protein Original Research Article American Journal of Experimental Agriculture, 4(7): 785-796, 2014 786 level. Chilling stress sequentially upregulated tomato CBF homolog, SlCBF1 and trehalose-6-phosphate synthase (SlTPS1). Based on the whole genome database of tomato, cis-elements potentially binding to ICE1 and CBF were located in upstream sequences in promoter regions of SlCBF1 and SlTPS1, respectively. Conclusion: Tomato ICE1 homolog mediates the expression of SlCBF1in a cold-stressinduced transcription factor cascade via binding to ICE1-specific cis-elements, leading to induction of cold tolerance by trehalose synthesis.

Overexpression of the AtSTK gene increases salt, PEG and ABA tolerance in Arabidopsis

AtSTK (At5g02800), which is a serine-threonine protein kinase gene of Arabidopsis thaliana, was cloned, and its function was studied. The study found that the overexpression of AtSTK could significantly improve the ability of A. thaliana to tolerate salt, PEG, and ABA stresses. RT-PCR analysis revealed that the expression of the AtSTK gene could be obviously induced by salt, PEG, and ABA. The examination of the physiological characteristics showed that the overexpression of AtSTK in Arabidopsis significantly reduced the plasma membrane permeability, significantly increased the proline content, and decreased the MDA content. These changes may reflect the physiological mechanisms through which AtSTK overexpression improves stress resistance in Arabidopsis. In addition, the overexpression of the AtSTK gene significantly antagonised the inhibitory effect of high concentrations of exogenous ABA on Arabidopsis seed germination. The subcellular localisation results showed that AtSTK is located in both the cytosol and the nucleus. The examination of its tissue-specific expression showed that AtSTK is expressed in various Arabidopsis tissues and is particularly strongly expressed in the vessels. The signalling pathway analysis indicated that AtSTK might transfer the salt stress signal in Arabidopsis through the MAPK pathway.

Overexpression of constitutively active mitogen activated protein kinase kinase 6 enhances tolerance to salt stress in rice

BackgroundSalinity is one of the most common abiotic stresses encountered by plants in the environment and transgenic approaches offer new opportunities to improve tolerance. The mitogen activated protein kinase (MAPK) kinase (MKK) is a key component of MAPK cascade that plays important roles in intra and extra cellular signaling in plants. In the present study, a MKK from rice (Oryza sativa), OsMKK6 was functionally characterized in salt stress by transforming its constitutively active form.FindingsOsMKK6 was made constitutively active by mutating serine and threonine to glutamic acid by site directed mutagenesis, and transformed in indica cultivar rice var. Pusa Basmati-1. The transgenic seedlings growing in 200 mM NaCl solution showed increased root/shoot length and weight, less chlorophyll beaching and higher MAPK activity compared to the wild types.ConclusionPresent work suggest role of OsMKK6 gene in salt stress signaling in rice.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-6-25) contains supplementary material, which is available to authorized users.

The overexpression of a maize mitogen‐activated protein kinase gene (ZmMPK5) confers salt stress tolerance and induces defence responses in tobacco

As sessile organisms, plants are exposed to potential dangers, including multiple biotic and abiotic stresses. The mitogen-activated protein kinase (MAPK) is a universal signalling pathways involved in these processes. A previous study showed that maize ZmMPK5 is induced by various stimuli at transcriptional and post-translational levels. In this study, ZmMPK5 was overexpressed in tobacco to further analyse its biological functions. Under salt and oxidative stresses, ZmMPK5-overexpressing lines displayed less severe damage and stronger growth phenotypes corresponding to a series of physiological changes. In addition, the transgenic lines accumulated less reactive oxygen species (ROS) and had higher levels of antioxidant enzyme activity and metabolites than wild-type (WT) plants following NaCl treatment. Quantitative RT-PCR revealed that the expression of ROS-related and stress-responsive genes was higher in transgenic plants than in WT plants. Furthermore, transgenic lines exhibited enhanced resistance to viral pathogens, and expressed constitutively higher transcript levels of pathogenesis-related genes, such as PR1a, PR4, PR5 and EREBP. Taken together, these results demonstrated that ZmMPK5 is involved in salt stress, oxidative stress and pathogen defence signalling pathways, and its function may be at least partly devoted to efficiently eliminating ROS accumulation under salt stress.

Salt-responsive ERF1 regulates reactive oxygen species-dependent signaling during the initial response to salt stress in rice.

Early detection of salt stress is vital for plant survival and growth. Still, the molecular processes controlling early salt stress perception and signaling are not fully understood. Here, we identified salt-responsive ERF1 (SERF1), a rice (Oryza sativa) transcription factor (TF) gene that shows a root-specific induction upon salt and hydrogen peroxide (H2O2) treatment. Loss of SERF1 impairs the salt-inducible expression of genes encoding members of a mitogen-activated protein kinase (MAPK) cascade and salt tolerance-mediating TFs. Furthermore, we show that SERF1-dependent genes are H2O2 responsive and demonstrate that SERF1 binds to the promoters of MAPK kinase kinase6 (MAP3K6), MAPK5, dehydration-responsive element bindinG2A (DREB2A), and zinc finger protein179 (ZFP179) in vitro and in vivo. SERF1 also directly induces its own gene expression. In addition, SERF1 is a phosphorylation target of MAPK5, resulting in enhanced transcriptional activity of SERF1 toward its direct target genes. In agreement, plants deficient for SERF1 are more sensitive to salt stress compared with the wild type, while constitutive overexpression of SERF1 improves salinity tolerance. We propose that SERF1 amplifies the reactive oxygen species-activated MAPK cascade signal during the initial phase of salt stress and translates the salt-induced signal into an appropriate expressional response resulting in salt tolerance.

마이크로어레이를 이용한 애기장대 AtERF71/HRE2 전사인자의 하위 유전자 분석

Arabidopsis AtERF71/HRE2, a transcription activator, is located in the nucleus and is involved in the signal transduction of low oxygen and osmotic stresses. In this study, microarray analysis using AtERF71/HRE2-overexpressing transgenic plants was performed to identify genes downstream of AtERF71/HRE2. A total of 161 different genes as well as AtERF71/HRE2 showed more than a twofold higher expression in AtERF71/HRE2-overexpressing transgenic plants compared with wild-type plants. Among the 161 genes, 24 genes were transcriptional regulators, such as transcription factors and DNA-binding proteins, based on gene ontology annotations, suggesting that AtERF71/HRE2 is an upstream transcription factor that regulates the activities of various downstream genes via these transcription regulators. RT-PCR analysis of 15 genes selected out of the 161 genes showed higher expression in AtERF71/HRE2-overexpressing transgenic plants, validating the microarray data. On the basis of Genevestigator database analysis, 51 genes among the 161 genes were highly expressed under low oxygen and/or osmotic stresses. RT-PCR analysis showed that the expression levels of three genes among the selected 15 genes increased under low oxygen stress and another three genes increased under high salt stress, suggesting that these genes might be downstream genes of AtERF71/HRE2 in low oxygen or high salt stress signal transduction. Microarray analysis results indicated that AtERF71/HRE2 might also be involved in the responses to other abiotic stresses and also in the regulation of plant developmental processes.

Soybean MAPK, GMK1 Is Dually Regulated by Phosphatidic Acid and Hydrogen Peroxide and Translocated to Nucleus during Salt Stress

Mitogen-activated protein kinase (MAPK) is activated by various biotic and abiotic stresses. Salt stress induces two well-characterized MAPK activating signaling molecules, phosphatidic acid (PA) via phospholipase D and phospholipase C, and reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. In our previous study, the activity of soybean MAPK, GMK1 was strongly induced within 5 min of 300 mM NaCl treatment and this early activity was regulated by PA. In this study, we focused on the regulation of GMK1 at the later stage of the salt stress, because its activity was strongly persistent for up to 30 min. H(2)O(2) activated GMK1 even in the presence of PA generation inhibitors, but GMK1 activity was greatly decreased in the presence of diphenyleneiodonium, an inhibitor of NADPH-oxidase after 5 min of the treatment. On the contrary, the n-butanol and neomycin reduced GMK1 activity within 5 min of the treatment. Thus, GMK1 activity may be sustained by H(2)O(2) 10 min after the treatment. Further, GMK1 was translocated into the nucleus 60 min after NaCl treatment. In the relationship between GMK1 and ROS generation, ROS generation was reduced by SB202190, a MAPK inhibitor, but was increased in protoplast overexpressing TESD-GMKK1. However, these effects were occurred at prolonged time of NaCl treatment. These data suggest that GMK1 indirectly regulates ROS generation. Taken together, we propose that soybean GMK1 is dually regulated by PA and H(2)O(2) at a time dependant manner and translocated to the nucleus by the salt stress signal.

A salt stress-activated mitogen-activated protein kinase in soybean is regulated by phosphatidic acid in early stages of the stress response

Salt stress inhibits plant growth and development and plants activate kinase-dependent survival pathways in response to salt stress. However, the role of soybean mitogenactivated protein kinases (MAPKs) in salt stress response has yet to be characterized. In this study, we found that salt stress activates Glycine max MAP kinase 1 (GMK1), a soybean MAPK. The activity of GMK1 induced with increasing salt concentrations, up to 300 mM NaCl, after 5 min of the treatment and was regulated by post-translational modification. We found that mastoparan, a heteromeric G-protein activator, also activated GMK1, and that n-butanol, a phospholipase D inhibitor, and neomycin, a phospholipase C inhibitor, inhibited its activity. Moreover, GMK1 activity was reduced by suramin, a heteromeric G-protein inhibitor, and by two inhibitors of phosphatidic acid (PA) generation after 5 min of 300 mM NaCl treatment. Endogenous PA levels were highest 5 min after induction of salt stress, and exogenous PA directly activated GMK1. From these data, we propose that salt stress signaling is transduced from heteromeric G-protein to GMK1 via phospholipases in the early stages of the response to salt stress in soybean.

Salt tolerance in soybean WF-7 is partially regulated by ABA and ROS signaling and involves withholding toxic Cl− ions from aerial tissues

Salt tolerance in plants is a complex trait involving multiple mechanisms. Understanding these mechanisms and their regulation will assist in developing novel strategies to engineer salt-tolerant crops. In the current study, we investigated salt-tolerant mechanisms in soybean (Glycine max) cultivar WF-7 in comparison to salt-sensitive Union. In vivo and in vitro salt assays demonstrated the salt tolerance of WF-7 at the seedling stage and during germination. After a 10-day 200 mM NaCl treatment, chlorophyll content in Union was reduced by 50 % compared to a 17 % reduction in WF-7. WF-7 was also less affected by abscisic acid (ABA) and NaCl during germination than Union. Upon ABA and NaCl treatment, the ABA-responsive genes SCOF1, ASN1, bZIP44, and AAPK1 are differentially expressed in WF-7 and Union seedlings. These results suggest that salt tolerance in WF-7 is in part regulated through an ABA-dependent pathway. In addition, following a 4-day 200 mM NaCl treatment, WF-7 produced more H₂O₂ than Union indicating the involvement of reactive oxygen species (ROS) in regulating salt tolerance in WF-7. Yet another mechanism WF-7 employs is withholding toxic chloride (Cl⁻) ions from aerial tissues. Following 200 mM NaCl treatment, Cl⁻ accumulation was mostly localized to the roots of WF-7. In contrast, most of the Cl⁻ in Union was transported into the stems and leaves. Taken together, our results demonstrated a role of ABA and ROS in regulating salt tolerance in WF-7, and the critical role of Cl⁻ in NaCl-induced mortality in soybean. Key message Withholding toxic Cl⁻ ions from leaves and, to a lesser extent, stems, confers salt tolerance to soybean WF-7. In addition, ABA and ROS may be involved in salt-stress signal transduction.

The jasmonate pathway mediates salt tolerance in grapevines.

Salt stress is a major constraint for many crop plants, such as the moderately salt-sensitive economically important fruit crop grapevine. Plants have evolved different strategies for protection against salinity and drought. Jasmonate signalling is a central element of both biotic and abiotic stress responses. To discriminate stress quality, there must be cross-talk with parallel signal chains. Using two grapevine cell lines differing in salt tolerance, the response of jasmonate ZIM/tify-domain (JAZ/TIFY) proteins (negative regulators of jasmonate signalling), a marker for salt adaptation Na+/H+ EXCHANGER (NHX1), and markers for biotic defence STILBENE SYNTHASE (StSy) and RESVERATROL SYNTHASE (RS) were analysed. It is shown that salt stress signalling shares several events with biotic defence including activity of a gadolinium-sensitive calcium influx channel (monitored by apoplastic alkalinization) and transient induction of JAZ/TIFY transcripts. Exogenous jasmonate can rescue growth in the salt-sensitive cell line. Suppression of jasmonate signalling by phenidone or aspirin blocks the induction of JAZ/TIFY transcripts. The rapid induction of RS and StSy characteristic for biotic defence in grapevine is strongly delayed in response to salt stress. In the salt-tolerant line, NHX1 is induced and the formation of reactive oxygen species, monitored as stress markers in the sensitive cell line, is suppressed. The data are discussed in terms of a model where salt stress signalling acts as a default pathway whose readout is modulated by a parallel signal chain triggered by biotic factors downstream of jasmonate signalling.

Comparative proteomic analysis of the Arabidopsis cbl1 mutant in response to salt stress

Many environmental stimuli, including light, biotic and abiotic stress factors, induce changes in cellular Ca(2+) concentrations in plants. Such Ca(2+) signatures are perceived by sensor molecules such as calcineurin B-like (CBL) proteins. AtCBL1, a member of the CBL family which is highly inducible by multiple stress signals, is known to function in the salt stress signal transduction pathway and to positively regulate the plant tolerance to salt. To shed light into the molecular mechanisms of the salt stress response mediated by AtCBL1, a two-dimensional DIGE proteomic approach was applied to identify the differentially expressed proteins in Arabidopsis wild-type and cbl1 null mutant plants in response to salt stress. Seventy-three spots were found altered in expression by least 1.2-fold and 50 proteins were identified by MALDI-TOF/TOF-MS, including some well-known and novel salt-responsive proteins. These proteins function in various processes, such as signal transduction, ROS scavenging, energy production, carbon fixation, metabolism, mRNA processing, protein processing and structural stability. Receptor for activated C kinase 1C (RACK1C, spot 715), a WD40 repeat protein, was up-regulated in the cbl1 null mutant, and two rack1c mutant lines showed decreased tolerance to salt stress, suggesting that RACK1C plays a role in salt stress resistance. In conclusion, our work demonstrated the advantages of the proteomic approach in studies of plant biology and identified candidate proteins in CBL1-mediated salt stress signaling network.

Expression pattern of wheat miRNAs under salinity stress and prediction of salt-inducible miRNAs targets

MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression by translational repression or transcript degradation. Thus far, a large number of miRNAs have been identified from model plant species and the quantity of miRNAs has been functionally characterized in diverse plants. However, the molecular characterizations of the conserved miRNAs are still largely elusive in wheat. In this study, 32 wheat miRNAs (TaMIRs) currently released in the Sanger miRBase (the microRNA database) were selected to evaluate the expression patterns under conditions of non-stress (CK) and salt stress treatment. Based on the analysis of semiquantitative RT-PCR and quantitative real qRT-PCR, TaMIR159a, TaMIR160, TaMIR167, TaMIR174, TaMIR399, TaMIR408, TaMIR11124 and TaMIR1133 were found to have responses to salinity stress, with an upregulated pattern under salt stress treatment. Based on a BLAST search against the NCBI GenBank database, the potential targets of the salt-inducible wheat miRNAs were predicted. Except for TaMIR399 not being identified to have the putative target genes, other salt-inducible TaMIRs were found to possess 2 to 7 putative target genes. Together, our results suggest that a subset of miRNAs are involved in the mediation of salt stress signaling responses in wheat via their roles on the regulation of acted target genes at post-transcriptional and translation levels.

Arabidopsis thaliana VDAC2 involvement in salt stress response pathway

Soil salinity seriously affects plants distribution and yield, while salt stress induces SOS genes, and voltage-dependent anion channels (VDAC) and a mitochondrial porin, are induced too. In this paper, phenotypes of AtVDAC2 transgenic lines and wild type (RLD) were analyzed. It was found that AtVDAC2 over-expressing transgenic plants were more sensitive to NaCl, and produced more H 2 O 2 in the NaCl treatment. Also, to find the inner reason, the salt overly sensitive gene 3 (SOS3) expression level was changed with the expression of AtVDAC2. So, it was conjectured that the signal of salt stress response was first sent to AtVDAC2, then AtVDAC2 expression improved, leading to the down-stream signals changes, such as accumulation of H 2 O 2 and improved expression of SOS3. So, it was found that in the over-expression of transgenic lines with AtVDAC2 up-regulation, SOS3 expression increased significantly, and in the inhibited-expressing lines, it was vice versa. In summary, AtVDAC2 was involved in salt stress signaling pathway, and it regulated SOS3 gene expression. Key words: Arabidopsis thaliana, voltage-dependent anion channels (VDAC), salt stress, signaling pathway.

Auxin modulation of salt stress signaling in Arabidopsis seed germination

Seed germination is an elaborate developmental process that is regulated through intricate signaling networks integrating diverse environmental cues into endogenous hormonal signaling pathways. Accumulating evidence in recent years supports the role of auxin in seed germination. Whereas the roles of gibberellic acid (GA) and abscisic acid (ABA) in the germination process have been studied extensively, how auxin modulates seed germination is largely unknown. We found that a membrane-bound NAC transcription factor NTM2 mediates the signaling crosstalk between auxin and salt stress via the IAA30 gene during seed germination in Arabidopsis. Germination of the NTM2-deficient ntm2-1 mutant seeds exhibited enhanced resistance to high salinity. However, the salt resistance was reduced in the ntm2-1 mutant overexpressing the IAA30 gene, which was induced by high salinity in a NTM2-dependent manner. Exogenous auxin treatment further suppressed the reduced germination rate of control seeds under high salinity. In contrast, the auxin effects disappeared in the ntm2-1 mutant. These observations indicate that NTM2 is a molecular link that incorporates auxin signal into salt stress signaling during seed germination, providing a role of auxin in modulating seed germination under high salinity.

Recent Updates on Salinity Stress in Rice: From Physiological to Molecular Responses

One-fifth of irrigated agriculture is negatively affected by high soil salinity. The expected population growth, over 9 billion by 2050, enhances the pressure for agricultural production in marginal saline lands. Rice (Oryza sativa L.), the staple food for more than half of the world's population, is the most salt-sensitive cereal. The need for salt-tolerant rice varieties able to cope with several other stress conditions obviously puts a lot of pressure on breeders who must better comprehend the physiology and genetic control of salt tolerance. In spite of several good reviews recently published, an integrated vision of current information on rice tolerance to salt stress has been lacking. Here we present the most recent data on the salinity effect on rice physiology and stress adaptation, including implications on growth regulation and reproductive development. We have included an inventory of salt tolerance donors available for breeding programs and a comprehensive survey of current work on QTL detection and cloning as well as marker-assisted selection to introgress favorable alleles into elite rice lines. A schematic view of the rice chromosomes on which salt tolerance QTLs and candidate genes are positioned is also included. Finally, we focus on the most promising candidate genes involved in salt stress response. There, we discuss the available knowledge on salt stress signaling and ion homeostasis, LEAs and other stress-induced proteins, genes with unknown function and transcription regulators as well as the present knowledge on the role of post-translational modifications on the modulation of the response to salinity in rice. We conclude by highlighting still missing clues that could help to design better salt tolerant varieties, and we evaluate the significance of the data presented for the future of rice breeding and sustainability of the culture in marginal saline soils.

THE ROLE OF PHOSPHOLIPASE C IN SIGNAL TRANSDUCTION DURING DROUGHT AND COLD STRESSES IN WHEAT (Triticum aestivum L.)

Environmental stresses are one of the major challenges to crop production. Molecular biologists have investigated these problems through the study of the genes whose expression could be changed in response to stress with the hypothesis that these genes may contribute to tolerance to these stresses. Zhu (2002) stated that salt and drought stress signal transduction consists of ionic and osmotic homeostasis signaling pathways, detoxification response pathways, and growth regulation pathways. Some phospholipids systems are activated by osmotic stress, generating a diverse array of messenger molecules, some of them function upstream of the osmotic stress-activated protein kinases. Also, ABA biosynthesis is regulated by osmotic stress. Both ABA dependent and independent osmotic stress signaling start with the modification of the expressed transcription factors, leading to the expression of early response transcriptional activators, which then activate downstream, stress tolerance effect or genes. Many cold responsive genes have been identified and characterized since the 1980’s. More recently DNA microarrays analysis using a 24 K GeneChips representing about 24000 Arabidopsis genes have been used to characterize changes in gene expression in response to cold treatment (Zhu et al., 2007). One study identified 655 genes cold upregulated and 284 as cold downregulated genes after chilling treatment (Lee et al., 2005). Many of the early cold responsive genes encode transcription factors that probably activate the genes that are induced after longer periods of exposure to the cold. Only one transcription factor was down regulated during early cold stress, suggesting that cold responses in plants are started by transcriptional activation and not by repression of genes. To investigate the interaction of proteins that may have signalling or regulatory function, protein-protein interaction was studied with yeast 2 hybrid analysis (Tardif et al., 2007). Phospholipases are sets of enzymes which have the ability to hydrolize phospholipids. They have different forms in plants, including phosphlipases C, D and A. Phospho-lipase C and D have important roles in signal transduction. The general structure of phospholipids consists of two fatty acyl chains esterified to a glycerol backbone at the sn-1 and sn-2 positions, and a phosphate at sn-3 position to which a variable head group (R) is attached. Phos-phatidylglycerol plays a specific role in photosynthesis (Sakurai et al., 2007) by helping in the organization and function of thylakoid membranes in plant cells. The first cell change during stress injury is an alteration in the structure and function of cell membranes. In many plants, alterations in lipids, particularly in phospholipids and sterols were very clear due to water stress (Liljenberg, 1992) or salt treatment (Kuiper, 1984). These alterations in the membrane seem to control the fluidity and the surrounding proteins, which influences membrane functions such as bilayer permeability carrier-mediated transport and the activity of membrane bound enzymes including ATPase activity (Surjus and Durand, 1996). In plants, G-proteins are involved in the regulation of ion channel and abscisic acid signalling, modulation of cell proliferation, and in many other processes such as seed germination, shoot and root growth, and stomatal regulation. Phospholipase C (PLC) has been shown to function as an intercellular effector molecule in plants for the α-subunit of pea heterotrimeric G-proteins whose activity is regulated by salinity stress (Misra et al., 2007). Gα protein interacts with pea phos-pholipase-C (PLC) at the calcium-binding domain (C2) leading to increasing GTPase activity, thus the signal transduction can be reduced by PLC association with the Gα-protein (Tuteja, 2007). The main objective of this study was to characterize the expression profiles of three full length cDNA clones from two phosphogyceride specific phospholipase C’s wheat genes under drought and cold stresses.

Analysis of Cytokinin Mutants and Regulation of Cytokinin Metabolic Genes Reveals Important Regulatory Roles of Cytokinins in Drought, Salt and Abscisic Acid Responses, and Abscisic Acid Biosynthesis

Cytokinins (CKs) regulate plant growth and development via a complex network of CK signaling. Here, we perform functional analyses with CK-deficient plants to provide direct evidence that CKs negatively regulate salt and drought stress signaling. All CK-deficient plants with reduced levels of various CKs exhibited a strong stress-tolerant phenotype that was associated with increased cell membrane integrity and abscisic acid (ABA) hypersensitivity rather than stomatal density and ABA-mediated stomatal closure. Expression of the Arabidopsis thaliana ISOPENTENYL-TRANSFERASE genes involved in the biosynthesis of bioactive CKs and the majority of the Arabidopsis CYTOKININ OXIDASES/DEHYDROGENASES genes was repressed by stress and ABA treatments, leading to a decrease in biologically active CK contents. These results demonstrate a novel mechanism for survival under abiotic stress conditions via the homeostatic regulation of steady state CK levels. Additionally, under normal conditions, although CK deficiency increased the sensitivity of plants to exogenous ABA, it caused a downregulation of key ABA biosynthetic genes, leading to a significant reduction in endogenous ABA levels in CK-deficient plants relative to the wild type. Taken together, this study provides direct evidence that mutual regulation mechanisms exist between the CK and ABA metabolism and signals underlying different processes regulating plant adaptation to stressors as well as plant growth and development.

Use of EcoTILLING to identify natural allelic variants of rice candidate genes involved in salinity tolerance

Rice is a salt-sensitive species with enormous genetic variation for salt tolerance hidden in its germplasm pool. The EcoTILLING technique allows us to assign haplotypes, thus reducing the number of accessions to be sequenced, becoming a cost-effective, time-saving and high-throughput method, ideal to be used in laboratories with limited financial resources. Aiming to find alleles associated with salinity tolerance, we are currently using the EcoTILLING technique to detect single nucleotide polymorphisms (SNPs) and small indels across 375 germplasm accessions representing the diversity available in domesticated rice. We are targeting several genes known to be involved in salt stress signal transduction (OsCPK17) or tolerance mechanisms (SalT). So far, we found a total of 15 and 23 representative SNPs or indels in OsCPK17 and SalT, respectively. These natural allelic variants are mostly located in 3′-untranslated region, thus opening a new path for studying their potential contribution to the regulation of gene expression and possible role in salt tolerance.

Molecular biology of cyanobacterial salt acclimation

High and changing salt concentrations represent major abiotic factors limiting the growth of microorganisms. During their long evolution, cyanobacteria have adapted to aquatic habitats with various salt concentrations. High salt concentrations in the medium challenge the cell with reduced water availability and high contents of inorganic ions. The basic mechanism of salt acclimation involves the active extrusion of toxic inorganic ions and the accumulation of compatible solutes, including sucrose, trehalose, glucosylglycerol, and glycine betaine. The kinetics of these physiological processes has been exceptionally well studied in the model Synechocystis 6803, leading to the definition of five subsequent phases in reaching a new salt acclimation steady state. Recent '-omics' technologies using the advanced model Synechocystis 6803 have revealed a comprehensive picture of the dynamic process of salt acclimation involving the differential expression of hundreds of genes. However, the mechanisms involved in sensing specific salt stress signals are not well resolved. In the future, analysis of cyanobacterial salt acclimation will be directed toward defining the functions of the many unknown proteins upregulated in salt-stressed cells, identifying specific salt-sensing mechanisms, using salt-resistant strains of cyanobacteria for the production of bioenergy, and applying cyanobacterial stress genes to improve the salt tolerance of sensitive organisms.

The woody plant poplar has a functionally conserved salt overly sensitive pathway in response to salinity stress

In Arabidopsis thaliana, the salt overly sensitive (SOS) pathway plays an essential role in maintaining ion homeostasis and conferring salt tolerance. Here we identified three SOS components in the woody plant Populus trichocarpa, designated as PtSOS1, PtSOS2 and PtSOS3. These putative SOS genes exhibited an overlapping but distinct expression pattern in poplar plants and the transcript levels of SOS1 and SOS2 were responsive to salinity stress. In poplar mesophyll protoplasts, PtSOS1 was specifically localized in the plasma membrane, whereas PtSOS2 was distributed throughout the cell, and PtSOS3 was predominantly targeted to the plasma membrane. Heterologous expression of PtSOS1, PtSOS2 and PtSOS3 could rescue salt-sensitive phenotypes of the corresponding Arabidopsis sos mutants, demonstrating that the Populus SOS proteins are functional homologues of their Arabidopsis counterpart. In addition, PtSOS3 interacted with, and recruited PtSOS2 to the plasma membrane in yeast and in planta. Reconstitution of poplar SOS pathway in yeast cells revealed that PtSOS2 and PtSOS3 acted coordinately to activate PtSOS1. Moreover, expression of the constitutively activated form of PtSOS2 partially complemented the sos3 mutant but not sos1, suggesting that PtSOS2 functions genetically downstream of SOS3 and upstream of SOS1. These results indicate a strong functional conservation of SOS pathway responsible for salt stress signaling from herbaceous to woody plants.