Evaluating physiological responses of plants to salinity stress.

BACKGROUND: Because soil salinity is a major abiotic constraint affecting crop yield, much research has been conducted to develop plants with improved salinity tolerance. Salinity stress impacts many aspects of a plant's physiology, making it difficult to study in toto Instead, it is more tractable to dissect the plant's response into traits that are hypothesized to be involved in the overall tolerance of the plant to salinity. SCOPE AND CONCLUSIONS: We discuss how to quantify the impact of salinity on different traits, such as relative growth rate, water relations, transpiration, transpiration use efficiency, ionic relations, photosynthesis, senescence, yield and yield components. We also suggest some guidelines to assist with the selection of appropriate experimental systems, imposition of salinity stress, and obtaining and analysing relevant physiological data using appropriate indices. We illustrate how these indices can be used to identify relationships amongst the proposed traits to identify which traits are the most important contributors to salinity tolerance. Salinity tolerance is complex and involves many genes, but progress has been made in studying the mechanisms underlying a plant's response to salinity. Nevertheless, several previous studies on salinity tolerance could have benefited from improved experimental design. We hope that this paper will provide pertinent information to researchers on performing proficient assays and interpreting results from salinity tolerance experiments.

A novel protein kinase involved in Na(+) exclusion revealed from positional cloning.

Salinity is a major abiotic stress which affects crop plants around the world, resulting in substantial loss of yield and millions of dollars of lost revenue. High levels of Na(+) in shoot tissue have many adverse effects and, crucially, yield in cereals is commonly inversely proportional to the extent of shoot Na(+) accumulation. We therefore need to identify genes, resistant plant cultivars and cellular processes that are involved in salinity tolerance, with the goal of introducing these factors into commercially available crops. Through the use of an Arabidopsis thaliana mapping population, we have identified a highly significant quantitative trait locus (QTL) linked to Na(+) exclusion. Fine mapping of this QTL identified a protein kinase (AtCIPK16), related to AtSOS2, that was significantly up-regulated under salt stress. Greater Na(+) exclusion was associated with significantly higher root expression of AtCIPK16, which is due to differences in the gene's promoter. Constitutive overexpression of the gene in Arabidopsis leads to plants with significant reduction in shoot Na(+) and greater salinity tolerance. amiRNA knock-downs of AtCIPK16 in Arabidopsis show a negative correlation between the expression levels of the gene and the amount of shoot Na(+) . Transgenic barley lines overexpressing AtCIPK16 show increased salinity tolerance.

A SOS3 homologue maps to HvNax4, a barley locus controlling an environmentally sensitive Na+ exclusion trait.

Genes that enable crops to limit Na(+) accumulation in shoot tissues represent potential sources of salinity tolerance for breeding. In barley, the HvNax4 locus lowered shoot Na(+) content by between 12% and 59% (g(-1) DW), or not at all, depending on the growth conditions in hydroponics and a range of soil types, indicating a strong influence of environment on expression. HvNax4 was fine-mapped on the long arm of barley chromosome 1H. Corresponding intervals of ∼200 kb, containing a total of 34 predicted genes, were defined in the sequenced rice and Brachypodium genomes. HvCBL4, a close barley homologue of the SOS3 salinity tolerance gene of Arabidopsis, co-segregated with HvNax4. No difference in HvCBL4 mRNA expression was detected between the mapping parents. However, genomic and cDNA sequences of the HvCBL4 alleles were obtained, revealing a single Ala111Thr amino acid substitution difference in the encoded proteins. The known crystal structure of SOS3 was used as a template to obtain molecular models of the barley proteins, resulting in structures very similar to that of SOS3. The position in SOS3 corresponding to the barley substitution does not participate directly in Ca(2+) binding, post-translational modifications or interaction with the SOS2 signalling partner. However, Thr111 but not Ala111 forms a predicted hydrogen bond with a neighbouring α-helix, which has potential implications for the overall structure and function of the barley protein. HvCBL4 therefore represents a candidate for HvNax4 that warrants further investigation.

Variation in salinity tolerance and shoot sodium accumulation in Arabidopsis ecotypes linked to differences in the natural expression levels of transporters involved in sodium transport.

Salinity tolerance can be attributed to three different mechanisms: Na+ exclusion from the shoot, Na+ tissue tolerance and osmotic tolerance. Although several key ion channels and transporters involved in these processes are known, the variation in expression profiles and the effects of these proteins on Na+ transport in different accessions of the same species are unknown. Here, expression profiles of the genes AtHKT1;1, AtSOS1, AtNHX1 and AtAVP1 are determined in four ecotypes of Arabidopsis thaliana. Not only are these genes differentially regulated between ecotypes, the expression levels of the genes can be linked to the concentration of Na+ in the plant. An inverse relationship was found between AtSOS1 expression in the root and total plant Na+ accumulation, supporting a role for AtSOS1 in Na+ efflux from the plant. Similarly, ecotypes with high expression levels of AtHKT1;1 in the root had lower shoot Na+ concentrations, due to the hypothesized role of AtHKT1;1 in retrieval of Na+ from the transpiration stream. The inverse relationship between shoot Na+ concentration and salinity tolerance typical of most cereal crop plants was not demonstrated, but a positive relationship was found between salt tolerance and levels of AtAVP1 expression, which may be related to tissue tolerance.

Sodium exclusion QTL associated with improved seedling growth in bread wheat under salinity stress.

Worldwide, dryland salinity is a major limitation to crop production. Breeding for salinity tolerance could be an effective way of improving yield and yield stability on saline-sodic soils of dryland agriculture. However, this requires a good understanding of inheritance of this quantitative trait. In the present study, a doubled-haploid bread wheat population (Berkut/Krichauff) was grown in supported hydroponics to identify quantitative trait loci (QTL) associated with salinity tolerance traits commonly reported in the literature (leaf symptoms, tiller number, seedling biomass, chlorophyll content, and shoot Na(+) and K(+) concentrations), understand the relationships amongst these traits, and determine their genetic value for marker-assisted selection. There was considerable segregation within the population for all traits measured. With a genetic map of 527 SSR-, DArT- and gene-based markers, a total of 40 QTL were detected for all seven traits. For the first time in a cereal species, a QTL interval for Na(+) exclusion (wPt-3114-wmc170) was associated with an increase (10%) in seedling biomass. Of the five QTL identified for Na(+) exclusion, two were co-located with seedling biomass (2A and 6A). The 2A QTL appears to coincide with the previously reported Na(+) exclusion locus in durum wheat that hosts one active HKT1;4 (Nax1) and one inactive HKT1;4 gene. Using these sequences as template for primer design enabled mapping of at least three HKT1;4 genes onto chromosome 2AL in bread wheat, suggesting that bread wheat carries more HKT1;4 gene family members than durum wheat. However, the combined effects of all Na(+) exclusion loci only accounted for 18% of the variation in seedling biomass under salinity stress indicating that there were other mechanisms of salinity tolerance operative at the seedling stage in this population. Na(+) and K(+) accumulation appear under separate genetic control. The molecular markers wmc170 (2A) and cfd080 (6A) are expected to facilitate breeding for salinity tolerance in bread wheat, the latter being associated with seedling vigour.

Arbuscular mycorrhizal inhibition of growth in barley cannot be attributed to extent of colonization, fungal phosphorus uptake or effects on expression of plant phosphate transporter genes.

Here, we used phosphorus-32 (32P) labelling in compartmented pots combined with quantitative real-time polymerase chain reaction (PCR) analysis of phosphate(Pi) transporter gene expression to investigate regulation of Pi uptake pathways in barley (Hordeum vulgare), an arbuscular mycorrhizal (AM) plant that does not show strong positive growth responses to colonization.Barley was colonized well by Glomus intraradices and poorly by Glomus geosporum,but both fungi induced significant and similar growth depressions compared with non mycorrhizal controls. The lack of correlation between per cent colonization and extent of growth depression suggests that the latter is not related to carbon drain to the fungus. The contribution of the AM Pi uptake pathway for the two fungi was, in general,related to per cent colonization and expression of the AM-inducible Pi transporter gene, HvPT8, but not to plant responsiveness. Glomus intraradices contributed 48%of total plant P whereas G. geosporum contributed very little.The growth depression in plants where the AM uptake pathway was functional suggests that the contribution of the direct Pi uptake pathway via root hairs and epidermis was decreased. This decrease was not correlated with downregulation of the epidermal-expressed Pi transporter genes, HvPT1 and HvPT2. We hypothesize post-transcriptional or post-translational control of this transport process by AM colonization.

Investigating glutamate receptor-like gene co-expression in Arabidopsis thaliana.

There is increasing evidence of the important roles of glutamate receptors (GLRs) in plant development and in adaptation to stresses. However, the studies of these putative ion channels, both in planta and in Xenopus oocytes, may have been limited by our lack of knowledge of possible GLR heteromer formation in plants. We have developed a modification of the single-cell sampling technique to investigate GLR co-expression, and thus potential heteromer formation, in single cells of Arabidopsis thaliana leaves. Micro-EXpression amplification (MEX) has allowed us to amplify gene transcripts from a single cell, enabling expression of up to 100 gene transcripts to be assayed. We measured, on average, the transcripts of five to six different AtGLRs in a single cell. However, no consistent patterns of co-expression or cell-type-specific expression were detected, except that cells sampled from the same plant showed similar expression profiles. The only discernible feature was the detection of AtGLR3.7 in every cell examined, an observation supported by GUS staining patterns in plants stably expressing promoter::uidA fusions. In addition, we found AtGLR3.7 expression in oocytes induces a Ba2+-, Ca2+- and Na+-permeable plasma membrane conductance.

Role of biosurfactant and ion channel-forming activities of syringomycin in transmembrane ion flux: a model for the mechanism of action in the plant-pathogen interaction.

Syringomycin is a necrosis-inducing lipopeptide toxin synthesized and secreted by the phytopathogen, Pseudomonas syringae pv. syringae. Although small quantities of syringomycin are known to activate a cascade of physiological events in plasma membranes, the mechanism of action of the phytotoxin has never been fully characterized. The objective of this study was to test the hypothesis that the primary mode of action of syringomycin is to form transmembrane pores that are permeable to cations. Accordingly, direct measurement of ion fluxes were performed using artificial bilayers. The hemolytic properties and surface activity of HPLC-purified syringomycin were quantified by use of an erythrocyte lysis assay and by the drop weight method. Assays were performed using syringomycin form SRE alone or a mixture containing all forms of the phytotoxin. At a threshold concentration of 500 ng/ml, syringomycin induced hemolysis by forming ion channels in membranes. Osmotic protection studies indicated a channel radius of between 0.6 and 1 nm. The ion channel-forming activity was insensitive and permeable to both monovalent and divalent cations, suggesting that syringomycin causes lysis of erythrocytes by colloid osmotic lysis. In addition, syringomycin, like other lipopeptide antibiotics, is a potent biosurfactant capable of lowering the interfacial tension of water to 31 mN/m. The critical micellar concentration of syringomycin was calculated to be 1.25 mg/ml and the gamma CMC was 33 mN/m. A model is presented depicting the mechanism of action of syringomycin in the plant-pathogen interaction. The model integrates known effects of the toxin on ion flux in plasma membranes with formation of ion channels and the consequential cascade of effects associated with cellular signalling.

Rapid pressure driven exocytosis-endocytosis cycle in a single plant cell. Capacitance measurements in aleurone protoplasts.

The whole-cell patch-clamp technique has been used to increase intracellular pressure via the pipette while monitoring changes in membrane capacitance (related to membrane area) in single aleurone protoplasts. Increased pressure increased membrane capacitance and diameter, upon the release of pressure both parameters returned to resting levels. Pressure also reversibly increased membrane conductance. Comparison between diameter and capacitance shows that the capacitance changes are due to changes in cell surface area. These results show that pressure can rapidly alter the balance between exo- and endocytosis. Pressure-induced changes in conductance are suggested to be due to insertion/withdrawal of channels with the exo-/endocytosed membrane.

A cytolytic delta-endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers.

In order to determine the mechanism of action of the 27 kDa mosquitocidal delta-endotoxin of Bacillus thuringiensis var. israelensis we have studied its effects on the conductance of planar lipid bilayers. The toxin formed cation-selective channels in the bilayers, permeable to K+ and Na+ but not to N-methylglucamine or Cl-, showing very fast, cooperative opening and closing. Channel opening was greatly reduced in the presence of divalent cations (Ca2+, Mg2+) and the effect was reversed when these ions were removed. These results are consistent with our proposal that B. thuringiensis toxins act by a mechanism of colloid-osmotic lysis.

Chronic hypoxemia: effects on developing nitrergic and dopaminergic amacrine cells.

PURPOSE: Very low birth weight and growth-restricted children have visual impairments including reduced contrast sensitivity, a parameter mediated in part by dopaminergic amacrine cells. The origin of these deficits is uncertain. In experimental fetal growth restriction, induced by placental insufficiency, the morphology and number of dopaminergic amacrine cells as identified by tyrosine hydroxylase staining were examined. In addition, the subclass of nitrergic amacrine cells was examined because nitric oxide released from nitric oxide synthase- containing neurons is proposed as a mediator of neurotoxicity and might contribute to the injury of dopaminergic amacrine cells in this situation. METHODS: Fetal sheep were subjected to 20 or 30 days of placental embolization leading to fetal hypoxemia, hypoglycemia, and growth restriction during the last third of gestation (term, approximately 147 days). Retinal tissue was prepared as wholemounts or cryostat sections and analyzed for retinal area, total number, density, somal size and cell process length of amacrine cells immunoreactive for tyrosine hydroxylase or nitric oxide synthase, and widths of retinal layers. Retinas from fetal sheep at 72, 96, 113, and 140 days' gestation and adults were collected for an ontogenetic study of tyrosine hydroxylase-immunoreactive neurons. RESULTS: In growth-restricted fetuses, the number of tyrosine hydroxylase-immunoreactive neurons and the total length of stained processes per cell were significantly reduced compared to control fetuses. The total number of neuronal nitric oxide synthase- containing neurons was not different between growth-restricted and control fetuses. The thickness of the inner retinal layers was reduced in hypoxemia. CONCLUSIONS: There is damage to tyrosine hydroxylase-immunoreactive amacrine cells during fetal chronic placental insufficiency. This damage might be involved in the altered retinal dopaminergic system observed in very low birth weight infants, some of whom are growth-restricted. Furthermore, a differential susceptibility of tyrosine hydroxylase-immunoreactive and neuronal nitric oxide synthase-containing amacrine cells to hypoxemic injury has been demonstrated. These observations add to the current hypothesis that neuronal nitric oxide synthase- containing neurons are resistant to hypoxemic injury and may be involved in mediating some of the neuronal damage that results from hypoxemic insults.

THE APPARENT WIDTH OF THE RHIZOSPHERE OF TRIFOLIUM SUBTERRANEUM L. FOR VESICULAR-ARBUSCULAR MYCORRHIZAL NFECTION: EFFECTS OF TIME AND OTHER FACTORS.

We use the mathematical model developed by Ferriss (1981) for the study of plant pathogens to calculate the apparent width of the rhizosphere (W) of Trifolium subterraneum L. for infection by propagules of vesicular-arbuscular mycorrhizal fungi in soil, examining the effects of wetting the soil for different times before planting and of harvesting at different times. The reasons for choosing this model rather than that of Gilligan (1979) are given. Propagule densities were determined by the most probable number (MPN) method at 28 d using the same host species as the test plant, thus including a factor in the calculations which is the product of the efficiency of the propagules to infect and the susceptibility of the roots. Values for W at 10 to 12 d were between 2.5 and 6.5 mm for mixed soil inoculum and between 8.9 and 13.2 mm for Glomus mosseae (Nicol & Gerd.) Gerdeman & Trappe. W increased with time, associated with an increase in the number of infections formed. Factors contributing to this include progressive germination of propagules following wetting the soil, growth of hyphae from greater distances to the root and formation of secondary infections. Wetting the soil before planting had the greatest effect on W in 4 d old plants. By 10 d, there was no effect, supporting the idea that most propagules are likely to have germinated by that time. The contribution of secondary infections is harder to determine, but is likely to be important from about 10 d onwards, resulting in overestimation of W. By 12 to 16 d, in experiments in which six plants were grown in 210 or 222 cm3 soil, the apparent volume of the rhizosphere plus root exceeded the volume of soil. Values of the apparent width of the rhizosphere in our experiments are therefore most reliable before the plants are 10 d old. If the MPN method is used to determine propagule densities at the same harvest times and under the same conditions as those used to collect other data, then the equations can be used to compare different fungi and environmental conditions with respect to infection of young plants.

Respiratory function in lambs after in utero treatment of lung hypoplasia by tracheal obstruction.

Tracheal obstruction (TO) stimulates growth of hypoplastic lungs in the fetus, but there is little knowledge of subsequent postnatal respiratory function. We have determined the effectiveness of TO in fetal sheep with existing lung hypoplasia in restoring postnatal respiratory function. Lung hypoplasia was induced by lung liquid drainage from 112 days of gestation to term ( approximately 148 days). We used an untreated group (ULH), a treated group (TLH) in which the trachea was obstructed for 10 days, and a control group. ULH lambs died within 4 h of birth. TLH lambs were hypoxic for the first week and were hypercapic at 2 days. Pulmonary diffusing capacity, gas volumes, and respiratory compliances were not different between control and TLH lambs. Minute ventilation was not different between the two groups; however, tidal volumes were lower and respiratory frequencies were higher in TLH lambs than in controls for 2 wk after birth. We conclude that 10 days of TO in the presence of initial lung hypoplasia prevents death at birth and returns most aspects of pulmonary function to normal by 1-2 wk after birth.

Asserting scientific authority. Cognitive development and adolescent legal rights.

The APA uses amicus briefs to communicate scientific knowledge to the legal system. There can be tension, however, between promoting the social good through law and the disinterested reporting of scientific data. This article examines this conflict by discussing two APA amicus briefs filed in the United States Supreme Court in cases involving adolescents' abortion rights. The Court has restricted adolescents' rights to make important life decisions in part because adolescents have been presumed to lack competence and maturity. The briefs argued that developmental theory and data confirm that adolescents and adults have equivalent decision-making capacities. The scientific arguments in the briefs, however, do not justify this assertion. Analysis of the briefs illuminates some dimensions describing the role of a scientific statement in a legal brief. These dimensions identify ways to limit scientific claims about the evidence at hand to avoid overstatement. The primary danger of overstatement is that it undermines psychology's claim to expert authority in assisting in the formation of law and the shaping of social institutions.

PIP: In response to US Supreme Court decisions that restricted the abortion rights of adolescents on the basis of their presumed lack of maturity and competence to make important life decisions, the American Psychological Association (APA) submitted amicus briefs in 1985 and 1987 claiming that adolescents have the same decision-making capabilities as adults. The APA briefs were in large part based on Piaget's cognitive developmental theory that formal operations, the final stage of cognitive development, begins at age 11-12 years and is completed by late adolescence. A review of these 2 briefs reveals a conflict between egalitarian political commitments and rigorous scientific standards. There are few data supporting the contention that adolescents and adults have equivalent decision-making skills; lacking are studies that actually compare adolescent and adult abilities. In the meager research the APA did cite in its brief, the difficulty of confirming a null hypothesis was underestimated, presumably due to a desire on the part of the APA to ensure that adolescents were not denied reproductive rights. The APA is now concerned, however, that such rejection of rigorous scientific method may undermine psychology's role as an expert authority in assisting in the formulation of law. To ensure the credibility of psychologists as scientists, the APA has developed a conceptual framework for social scientific claims in legal advocacy. Briefs must go beyond a mere digest of the available literature to provide a construal of the meaning of this research. The argument should contain a description of all events held to be relevant to a judicial decision, claims about the scientific status of that description, and a case built on theory and evidence from science to defend the claim regarding scientific status.

Techniques for studying ion channels: an introduction.

In this Introduction to the Special Issue on Ion Channels in Plants, the techniques used to study ion channels are reviewed. In particular, the basic approaches used for the electrophysiological study of ion channels are explained, from voltage clamping membranes and observing current changes over time, to the construction of current-voltage relations and the use of tail current protocols to determine channel selectivity. This is done for whole-cell and single channel records.

Ca2+-independent and Ca2+/GTP-binding protein-controlled exocytosis in a plant cell.

Exocytosis allows the release of secretory products and the delivery of new membrane material to the plasma membrane. So far, little is known about the underlying molecular mechanism and its control in plant cells. We have used the whole-cell patch-clamp technique to monitor changes in membrane capacitance to study exocytosis in barley aleurone protoplasts. To investigate the involvement of Ca2+ and GTP-binding proteins in exocytosis, protoplasts were dialyzed with very low (<2 nM) and high (1 microM) free Ca2+ and nonhydrolyzable guanine nucleotides guanosine 5'-gamma-thio]triphosphate (GTP[gammaS]) or guanosine 5'-[beta-thio]diphosphate (GDP[betaS]). With less than 2 nM cytoplasmic free Ca2+, the membrane capacitance increased significantly over 20 min. This increase was not altered by GTP[gammaS] or GDP[betaS]. In contrast, dialyzing protoplasts with 1 microM free Ca2+ resulted in a large increase in membrane capacitance that was slightly reduced by GTP[gammaS] and strongly inhibited by GDP[betaS]. We conclude that two exocytotic pathways exist in barley aleurone protoplasts: one that is Ca2+-independent and whose regulation is currently not known and another that is stimulated by Ca2+ and modulated by GTP-binding proteins. We suggest that Ca2+-independent exocytosis may be involved in cell expansion in developing protoplasts. Ca2+-stimulated exocytosis may play a role in gibberellic acid-stimulated alpha-amylase secretion in barley aleurone and, more generally, may be involved in membrane resealing in response to cell damage.