Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding,salicylic acid treatment,and salinity stress in safflower,Carthamus tinctorius

Safflower (Carthamus tinctorius L.) serves as a reference dicot for investigation of defence mechanisms in Asteraceae due to abundant secondary metabolites and high resistance/tolerance to environmental stresses. In plants, phenylpropanoid and flavonoid pathways are considered as two central defence signalling cascades in stress conditions. Here, we describe the isolation of two major genes in these pathways, CtPAL (phenylalanine ammonia-lyase) and CtCHS (chalcone synthase) in safflower along with monitoring their expression profiles in different stress circumstances. The aa (amino acid) sequence of isolated region of CtPAL possesses the maximum identity up to 96% to its orthologue in Cynara scolymus, while that of CtCHS retains the highest identity to its orthologue in Callistephus chinensis up to 96%. Experiments for gene expression profiling of CtPAL and CtCHS were performed after the treatment of seedlings with 0.1 and 1 mM SA (salicylic acid), wounding and salinity stress. The results of semi-quantitative RT–PCR revealed that both CtPAL and CtCHS genes are further responsive to higher concentration of SA with dissimilar patterns. Regarding wounding stress, CtPAL gets slightly induced upon injury at 3 hat (hours after treatment) (hat), whereas CtCHS gets greatly induced at 3 hat and levels off gradually afterward. Upon salinity stress, CtPAL displays a similar expression pattern by getting slightly induced at 3 hat, but CtCHS exhibits a biphasic expression profile with two prominent peaks at 3 and 24 hat. These results substantiate the involvement of phenylpropanoid and particularly flavonoid pathways in safflower during wounding and especially salinity stress.     

The Pathway and Regulation of Salicylic Acid Biosynthesis in Probenazole-Treated <Emphasis Type="Italic">Arabidopsis</Emphasis>

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide) is a highly effective chemical inducer of systemic-acquired resistance (SAR). It has been used widely to protect rice plants against the rice blast fungus Magnaporthe grisea. Previous studies have shown that PBZ induces SAR through enhanced accumulation of salicylic acid (SA). Plants synthesize SA by either a pathway that uses phenylalanine as substrate or another that involves isochorismate. To clarify how SA is produced in PBZ-treated Arabidopsis, we examined the expression patterns and enzyme activities of phenylalanine ammonia lyase (PAL) and isochorismate synthase (ICS), which are the main components of the phenylalanine and isochorismate pathways, respectively. PBZ exposure significantly improved the accumulation of SA and increased ICS activity. In the sid2–2 mutant, which has a defect in ICS1, PBZ had no effect on the level of endogenous SA or activity of ICS. In contrast, PAL activity and the expression of most PAL genes were down-regulated by such treatment in wild-type plants. These results suggest that SA is mainly synthesized via the ICS-mediated pathway in Arabidopsis.     

Biosynthesis of salicylic acid in plants

Salicylic acid (SA) is an important signal molecule in plants. Two pathways of SA biosynthesis have been proposed in plants. Biochemical studies using isotope feeding have suggested that plants synthesize SA from cinnamate produced by the activity of phenylalanine ammonia lyase (PAL). Silencing of PAL genes in tobacco or chemical inhibition of PAL activity in Arabidopsis, cucumber and potato reduces pathogen-induced SA accumulation. Genetic studies, on the other hand, indicate that the bulk of SA is produced from isochorismate. In bacteria, SA is synthesized from chorismate through two reactions catalyzed by isochorismate synthase (ICS) and isochorismate pyruvate lyase (IPL). Arabidopsis contains two ICS genes but has no gene encoding proteins similar to the bacterial IPL. Thus, how SA is synthesized in plants is not fully elucidated. Two recently identified Arabidopsis genes, PBS3 and EPS1, are important for pathogen-induced SA accumulation. PBS3 encodes a member of the acyl-adenylate/thioester-forming enzyme family and EPS1 encodes a member of the BAHD acyltransferase superfamily. PBS3 and EPS1 may be directly involved in the synthesis of an important precursor or regulatory molecule for SA biosynthesis. The pathways and regulation of SA biosynthesis in plants may be more complicated than previously thought.Key words: salicylic acid biosynthesis, isochorismate synthase, phenylalanine ammonia lyase     

Predicting future community-level ocular Chlamydia trachomatis infection prevalence using serological,clinical, molecular,and geospatial data

Trachoma is an infectious disease characterized by repeated exposures to Chlamydia trachomatis (Ct) that may ultimately lead to blindness. Efficient identification of communities with high infection burden could help target more intensive control efforts. We hypothesized that IgG seroprevalence in combination with geospatial layers, machine learning, and model-based geostatistics would be able to accurately predict future community-level ocular Ct infections detected by PCR. We used measurements from 40 communities in the hyperendemic Amhara region of Ethiopia to assess this hypothesis. Median Ct infection prevalence among children 0–5 years old increased from 6% at enrollment, in the context of recent mass drug administration (MDA), to 29% by month 36, following three years without MDA. At baseline, correlation between seroprevalence and Ct infection was stronger among children 0–5 years old (ρ = 0.77) than children 6–9 years old (ρ = 0.48), and stronger than the correlation between active trachoma and Ct infection (0-5y ρ = 0.56; 6-9y ρ = 0.40). Seroprevalence was the strongest concurrent predictor of infection prevalence at month 36 among children 0–5 years old (cross-validated R² = 0.75, 95% CI: 0.58–0.85), though predictive performance declined substantially with increasing temporal lag between predictor and outcome measurements. Geospatial variables, a spatial Gaussian process, and stacked ensemble machine learning did not meaningfully improve predictions. Serological markers among children 0–5 years old may be an objective tool for identifying communities with high levels of ocular Ct infections, but accurate, future prediction in the context of changing transmission remains an open challenge.     

Mass Treatment with Azithromycin for Trachoma: When Is One Round Enough? Results from the PRET Trial in The Gambia

Background

The World Health Organization has recommended three rounds of mass drug administration (MDA) with antibiotics in districts where the prevalence of follicular trachoma (TF) is ≥10% in children aged 1–9 years, with treatment coverage of at least 80%. For districts at 5–10% TF prevalence it was recommended that TF be assessed in 1–9 year olds in each community within the district, with three rounds of MDA provided to any community where TF≥10%. Worldwide, over 40 million people live in districts whose TF prevalence is estimated to be between 5 and 10%. The best way to treat these districts, and the optimum role of testing for infection in deciding whether to initiate or discontinue MDA, are unknown.

Methods

In a community randomized trial with a factorial design, we randomly assigned 48 communities in four Gambian districts, in which the prevalence of trachoma was known or suspected to be above 10%, to receive annual mass treatment with expected coverage of 80–89% (“Standard”), or to receive an additional visit in an attempt to achieve coverage of 90% or more (“Enhanced”). The same 48 communities were randomised to receive mass treatment annually for three years (“3×”), or to have treatment discontinued if Chlamydia trachomatis (Ct) infection was not detected in a sample of children in the community after mass treatment (stopping rule(“SR”)). Primary outcomes were the prevalence of TF and of Ct infection in 0–5 year olds at 36 months.

Results

The baseline prevalence of TF and of Ct infection in the target communities was 6.5% and 0.8% respectively. At 36 months the prevalence of TF was 2.8%, and that of Ct infection was 0.5%. No differences were found between the arms in TF or Ct infection prevalence either at baseline (Standard-3×: TF 5.6%, Ct 0.7%; Standard-SR: TF 6.1%, Ct 0.2%; Enhanced-3×: TF 7.4%, Ct 0.9%; and Enhanced-SR: TF 6.2%, Ct 1.2%); or at 36 months (Standard-3×: TF 2.3%, Ct 1.0%; Standard-SR TF 2.5%, Ct 0.2%; Enhanced-3× TF 3.0%, Ct 0.2%; and Enhanced-SR TF 3.2%, Ct 0.7% ). The implementation of the stopping rule led to treatment stopping after one round of MDA in all communities in both SR arms. Mean treatment coverage of children aged 0–9 in communities randomised to standard treatment was 87.7% at baseline and 84.8% and 88.8% at one and two years, respectively. Mean coverage of children in communities randomized to enhanced treatment was 90.0% at baseline and 94.2% and 93.8% at one and two years, respectively. There was no evidence of any difference in TF or Ct prevalence at 36 months resulting from enhanced coverage or from one round of MDA compared to three.

Conclusions

The Gambia is close to the elimination target for active trachoma. In districts prioritised for three MDA rounds, one round of MDA reduced active trachoma to low levels and Ct infection was not detectable in any community. There was no additional benefit to giving two further rounds of MDA. Programmes could save scarce resources by determining when to initiate or to discontinue MDA based on testing for Ct infection, and one round of MDA may be all that is necessary in some settings to reduce TF below the elimination threshold.     

Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana

Salicylic acid (SA) is an important signal involved in the activation of defence responses against abiotic and biotic stress. In tobacco, benzoic acid or glucosyl benzoate were proposed to be precursors of SA. This is in sharp contrast with studies in Arabidopsis thaliana, where SA derives from isochorismate. We have determined the importance of isochorismate for SA biosynthesis in Nicotiana benthamiana using virus-induced gene silencing of the isochorismate synthase (ICS) gene. Plants with silenced ICS expression do not accumulate SA after exposure to UV or to pathogen stress. Plants with silenced ICS expression also exhibit strongly decreased levels of phylloquinone, a product of isochorismate. Our data provide evidence for an isochorismate-derived synthesis of SA in N. benthamiana.     

Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca²⁺ or Mg²⁺ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.     

Synchronous Effects of Temperature, Hydrostatic Pressure, and Salinity on Growth, Phospholipid Profiles, and Protein Patterns of Four Halomonas Species Isolated from Deep-Sea Hydrothermal- Vent and Sea Surface Environments

Four strains of euryhaline bacteria belonging to the genus Halomonas were tested for their response to a range of temperatures (2, 13, and 30°C), hydrostatic pressures (0.1, 7.5, 15, 25, 35, 45, and 55 MPa), and salinities (4, 11, and 17% total salts). The isolates were psychrotolerant, halophilic to moderately halophilic, and piezotolerant, growing fastest at 30°C, 0.1 MPa, and 4% total salts. Little or no growth occurred at the highest hydrostatic pressures tested, an effect that was more pronounced with decreasing temperatures. Growth curves suggested that the Halomonas strains tested would grow well in cool to warm hydrothermal-vent and associated subseafloor habitats, but poorly or not at all under cold deep-sea conditions. The intermediate salinity tested enhanced growth under certain high-hydrostatic-pressure and low-temperature conditions, highlighting a synergistic effect on growth for these combined stresses. Phospholipid profiles obtained at 30°C indicated that hydrostatic pressure exerted the dominant control on the degree of lipid saturation, although elevated salinity slightly mitigated the increased degree of lipid unsaturation caused by increased hydrostatic pressure. Profiles of cytosolic and membrane proteins of Halomonas axialensis and H. hydrothermalis performed at 30°C under various salinities and hydrostatic pressure conditions indicated several hydrostatic pressure and salinity effects, including proteins whose expression was induced by either an elevated salinity or hydrostatic pressure, but not by a combination of the two. The interplay between salinity and hydrostatic pressure on microbial growth and physiology suggests that adaptations to hydrostatic pressure and possibly other stresses may partially explain the euryhaline phenotype of members of the genus Halomonas living in deep-sea environments.     

Effects of wounding and salicylic acid on hydroxycinnamoylmalic acids in Thunbergia alata

We describe the identification of three phenolic compounds (caffeoylmalic, feruloylmalic and p-coumaroylmalic acids) in the leaves of Thunbergia alata. Caffeoylmalic and feruloylmalic acids represent the majority of all the derivatives of hydroxycinnamic acid in this plant. Elicitation with 5 mM of salicylic acid (SA) after wounding produces an intense necrotic reaction which reaches a peak after 24 h. This reaction is much less intense when the leaves are subjected to wounding alone. HPLC analysis of the above three acids in leaf samples taken 24 h after treatment shows that the concentration of caffeoylmalic acid increases in the case of both wounding and SA treatment. The level of feruloylmalic acid increases principally in response to wounding while the concentration of p-coumaroylmalic acid increases essentially following elicitation by SA. The accumulation of these three compounds occurs not only in directly treated leaves, but also in untreated leaves situated above treated leaves (systemic accumulation). Wounding and SA produce a greater local accumulation of feruloyl and p-coumaroylmalic malic acid, respectively. For caffeoylmalic acid, wounding produces the greatest local accumulation whereas in response to SA, systemic accumulation of this compound is greater. Plants aged 2 months are more sensitive than younger plants to these two stress treatments.     

Central role of salicylic acid in resistance of safflower (Carthamus tinctorius L.) against salinity

The effects of salicylic acid (SA) on growth parameters and enzyme activities were investigated in salt-stressed safflower (Carthamus tinctorius L.). Twenty-five days after sowing, seedlings were treated with NaCl (0, 100, and 200?mM) and SA (1?mM), and were harvested at 21 days after treatments. Results showed that some growth parameters decreased under salinity, while malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) content, phenolic compounds, and some enzyme activities increased. SA application increased some growth parameters, MDA and H₂O₂ content, and enzyme activities except catalase (CAT), which was different from the other enzymes and SA significantly reduced CAT activity in plants. These results suggest that SA-induced tolerance to salinity may be related to regulation of antioxidative responses and H₂O₂ level. Our study suggested that the resistant safflower can direct reactive oxygen species from a threat to an opportunity by using SA. Therefore, exogenous application of SA played this role through regulation of the antioxidant system.     

Vitamin K1 accumulation in tobacco plants overexpressing bacterial genes involved in the biosynthesis of salicylic acid

Phylloquinone (Vitamin K(1)) is an essential component of the photosynthetic electron transfer. As isochorismate is required for the biosynthesis of Vitamin K(1), isochorismate synthase (ICS) activity is expected to be present in all green plants. In bacteria salicylic acid (SA) is synthesized via a two step pathway involving ICS and isochorismate pyruvate lyase (IPL). The effect of the introduction in tobacco plants of the bacterial ICS and IPL genes on the endogenous isochorismate pathway was investigated. Transgenic tobacco plants in which IPL was targeted to the chloroplast suffered severe growth retardation and had low Vitamin K(1) content. Probably because isochorismate was channeled towards SA production, the plants were no longer able to produce normal levels of Vitamin K(1). Transgenic tobacco plants in which the bacterial ICS was present in the chloroplast showed higher Vitamin K(1) contents than wild type plants.     

Inactivation of Enteric Adenovirus and Feline Calicivirus by Chlorine Dioxide

Chlorine dioxide (ClO₂) inactivation experiments were conducted with adenovirus type 40 (AD40) and feline calicivirus (FCV). Experiments were carried out in buffered, disinfectant demand-free water under high- and low-pH and -temperature conditions. Ct values (the concentration of ClO₂ multiplied by contact time with the virus) were calculated directly from bench-scale experiments and from application of the efficiency factor Hom (EFH) model. AD40 Ct ranges for 4-log inactivation (Ct_99.99%) at 5°C were >0.77 to <1.53 mg/liter × min and >0.80 to <1.59 mg/liter × min for pH 6 and 8, respectively. For 15°C AD40 experiments, >0.49 to <0.74 mg/liter × min and <0.12 mg/liter × min Ct_99.99% ranges were observed for pH 6 and 8, respectively. FCV Ct_99.99% ranges for 5°C experiments were >20.20 to <30.30 mg/liter × min and >0.68 mg/liter × min for pH 6 and 8, respectively. For 15°C FCV experiments, Ct_99.99% ranges were >4.20 to <6.72 and <0.18 mg/liter × min for pH 6 and 8, respectively. Viral inactivation was higher at pH 8 than at pH 6 and at 15°C than at 5°C. Comparison of Ct values and inactivation curves demonstrated that the EFH model described bench-scale experiment data very well. Observed bench-scale Ct_99.99% ranges and EFH model Ct_99.99% values demonstrated that FCV is more resistant to ClO₂ than AD40 for the conditions studied. U.S. Environmental Protection Agency guidance manual Ct_99.99% values are higher than Ct_99.99% values calculated from bench-scale experiments and from EFH model application.     

Effect of Salicylic Acid Formulations on Induced Plant Defense against Cassava Anthracnose Disease

This study was to investigate defense mechanisms on cassava induced by salicylic acid formulation (SA) against anthracnose disease. Our results indicated that the SA could reduce anthracnose severity in cassava plants up to 33.3% under the greenhouse condition. The β-1,3-glucanase and chitinase enzyme activities were significantly increased at 24 hours after inoculation (HAI) and decrease at 48 HAI after Colletotrichum gloeosporioides challenge inoculation, respectively, for cassava treated with SA formulation. Synchrotron radiation–based Fourier-transform infrared microspectroscopy spectra revealed changes of the C=H stretching vibration (3,000–2,800 cm⁻¹), pectin (1,740–1,700 cm⁻¹), amide I protein (1,700–1,600 cm⁻¹), amide II protein (1,600–1,500 cm⁻¹), lignin (1,515 cm⁻¹) as well as mainly C–O–C of polysaccharides (1,300–1,100 cm⁻¹) in the leaf epidermal and mesophyll tissues treated with SA formulations, compared to those treated with fungicide carbendazim and distilled water after the challenged inoculation with C. gloeosporioides. The results indicate that biochemical changes in cassava leaf treated with SA played an important role in the enhancement of structural and chemical defense mechanisms leading to reduced anthracnose severity.     

High temperatures change the perspective: Integrating hormonal responses in citrus plants under co‐occurring abiotic stress conditions

Plants growing in the field are subjected to multiple stress factors acting simultaneously. Abnormally high temperatures are expected to affect wild plants and crops in the next years due to global warming. In this work, we have studied physiological, hormonal and molecular responses of the citrus rootstock, Carrizo citrange (Poncirus trifoliata L. Raf. × Citrus sinensis L. Osb.) subjected to wounding or high salinity occurring individually or in combination with heat stress. According to our results, combination of high salinity and heat stress aggravated the negative effects of salt intoxication in Carrizo. The high transpiration rate caused by high temperatures counteracted physiological responses of plants to salt stress and increased Cl^? intake in leaves. In addition, 12‐oxo‐phytodienoic acid accumulated specifically under combination of wounding and heat stress, whereas at low temperatures, wounded plants accumulated jasmonic acid (JA) and JA‐isoleucine (JA‐Ile). Moreover, an antagonism between salicylic acid (SA) and JA was observed, and wounded plants subjected to high temperatures did not accumulate JA nor JA‐Ile whereas SA levels increased (via isochorismate synthase biosynthetic pathway). Wounded plants did not accumulate abscisic acid (ABA) but its catabolite phaseic acid. This could act as a signal for the upregulation of (ABA)‐RESPONSIVE ELEMENT (ABRE)‐BINDING TRANSCRIPTION FACTOR 2 (CsAREB2) and RESPONSIVE TO DISSECATION 22 (CsRD22) in an ABA‐independent way. This work uncovers some mechanisms that explain Carrizo citrange tolerance to high temperatures together with different hormonal signals in response to specific stresses. It is suggested that co‐occurring abiotic stress conditions can modify (either enhance or reduce) the hormonal response to modulate specific responses.     

Chilling- and Freezing- Induced Alterations in Cytosine Methylation and Its Association with the Cold Tolerance of an Alpine Subnival Plant,Chorispora bungeana

Chilling (0–18°C) and freezing (<0°C) are two distinct types of cold stresses. Epigenetic regulation can play an important role in plant adaptation to abiotic stresses. However, it is not yet clear whether and how epigenetic modification (i.e., DNA methylation) mediates the adaptation to cold stresses in nature (e.g., in alpine regions). Especially, whether the adaptation to chilling and freezing is involved in differential epigenetic regulations in plants is largely unknown. Chorispora bungeana is an alpine subnival plant that is distributed in the freeze-thaw tundra in Asia, where chilling and freezing frequently fluctuate daily (24 h). To disentangle how C. bungeana copes with these intricate cold stresses through epigenetic modifications, plants of C. bungeana were treated at 4°C (chilling) and -4°C (freezing) over five periods of time (0–24 h). Methylation-sensitive amplified fragment-length polymorphism markers were used to investigate the variation in DNA methylation of C. bungeana in response to chilling and freezing. It was found that the alterations in DNA methylation of C. bungeana largely occurred over the period of chilling and freezing. Moreover, chilling and freezing appeared to gradually induce distinct DNA methylation variations, as the treatment went on (e.g., after 12 h). Forty-three cold-induced polymorphic fragments were randomly selected and further analyzed, and three of the cloned fragments were homologous to genes encoding alcohol dehydrogenase, UDP-glucosyltransferase and polygalacturonase-inhibiting protein. These candidate genes verified the existence of different expressive patterns between chilling and freezing. Our results showed that C. bungeana responded to cold stresses rapidly through the alterations of DNA methylation, and that chilling and freezing induced different DNA methylation changes. Therefore, we conclude that epigenetic modifications can potentially serve as a rapid and flexible mechanism for C. bungeana to adapt to the intricate cold stresses in the alpine areas.     

Tolerance of Hordeum marinum accessions to O2 deficiency, salinity and these stresses combined

Background and Aims

When root-zone O₂ deficiency occurs together with salinity, regulation of shoot ion concentrations is compromised even more than under salinity alone. Tolerance was evaluated amongst 34 accessions of Hordeum marinum, a wild species in the Triticeae, to combined salinity and root-zone O₂ deficiency. Interest in H. marinum arises from the potential to use it as a donor for abiotic stress tolerance into wheat.

Methods

Two batches of 17 H. marinum accessions, from (1) the Nordic Gene Bank and (2) the wheat belt of Western Australia, were exposed to 0·2 or 200 mol m⁻³ NaCl in aerated or stagnant nutrient solution for 28–29 d. Wheat (Triticum aestivum) was included as a sensitive check species. Growth, root porosity, root radial O₂ loss (ROL) and leaf ion (Na⁺, K⁺, Cl⁻) concentrations were determined.

Key Results

Owing to space constraints, this report is focused mainly on the accessions from the Nordic Gene Bank. The 17 accessions varied in tolerance; relative growth rate was reduced by 2–38 % in stagnant solution, by 8–42 % in saline solution (aerated) and by 39–71 % in stagnant plus saline treatment. When in stagnant solution, porosity of adventitious roots was 24–33 %; salinity decreased the root porosity in some accessions, but had no effect in others. Roots grown in stagnant solution formed a barrier to ROL, but variation existed amongst accessions in apparent barrier ‘strength’. Leaf Na⁺ concentration was 142–692 µmol g⁻¹ d. wt for plants in saline solution (aerated), and only increased to 247–748 µmol g⁻¹ d. wt in the stagnant plus saline treatment. Leaf Cl⁻ also showed only small effects of stagnant plus saline treatment, compared with saline alone. In comparison with H. marinum, wheat was more adversely affected by each stress alone, and particularly when combined; growth reductions were greater, adventitious root porosity was 21 %, it lacked a barrier to ROL, leaf K⁺ declined to lower levels, and leaf Na⁺ and Cl⁻ concentrations were 3·1–9-fold and 2·8–6-fold higher, respectively, in wheat.

Conclusions

Stagnant treatment plus salinity reduced growth more than salinity alone, or stagnant alone, but some accessions of H. marinum were still relatively tolerant of these combined stresses, maintaining Na⁺ and Cl⁻ ‘exclusion’ even in an O₂-deficient, saline rooting medium.Key words: Aerenchyma, combined salinity and waterlogging, leaf Cl⁻, leaf K⁺, leaf Na⁺, radial O₂ loss, salt tolerance, salinity–waterlogging interaction, sea barleygrass, waterlogging tolerance, wheat, wild Triticeae