Plant salt tolerance: adaptations in halophytes

Background Most of the water on Earth is seawater, each kilogram of which contains about 35 g of salts, and yet most plants cannot grow in this solution; less than 0·2 % of species can develop and reproduce with repeated exposure to seawater. These ‘extremophiles’ are called halophytes.Scope Improved knowledge of halophytes is of importance to understanding our natural world and to enable the use of some of these fascinating plants in land re-vegetation, as forages for livestock, and to develop salt-tolerant crops. In this Preface to a Special Issue on halophytes and saline adaptations, the evolution of salt tolerance in halophytes, their life-history traits and progress in understanding the molecular, biochemical and physiological mechanisms contributing to salt tolerance are summarized. In particular, cellular processes that underpin the ability of halophytes to tolerate high tissue concentrations of Na⁺ and Cl⁻, including regulation of membrane transport, their ability to synthesize compatible solutes and to deal with reactive oxygen species, are highlighted. Interacting stress factors in addition to salinity, such as heavy metals and flooding, are also topics gaining increased attention in the search to understand the biology of halophytes.Conclusions Halophytes will play increasingly important roles as models for understanding plant salt tolerance, as genetic resources contributing towards the goal of improvement of salt tolerance in some crops, for re-vegetation of saline lands, and as ‘niche crops’ in their own right for landscapes with saline soils.     

Reptile freeze tolerance: metabolism and gene expression

Terrestrially hibernating reptiles that live in seasonally cold climates need effective strategies of cold hardiness to survive the winter. Use of thermally buffered hibernacula is very important but when exposure to temperatures below 0 degrees C cannot be avoided, either freeze avoidance (supercooling) or freeze tolerance strategies can be employed, sometimes by the same species depending on environmental conditions. Several reptile species display ecologically relevant freeze tolerance, surviving for extended times with 50% or more of their total body water frozen. The use of colligative cryoprotectants by reptiles is poorly developed but metabolic and enzymatic adaptations providing anoxia tolerance and antioxidant defense are important aids to freezing survival. New studies using DNA array screening are examining the role of freeze-responsive gene expression. Three categories of freeze responsive genes have been identified from recent screenings of liver and heart from freeze-exposed (5h post-nucleation at -2.5 degrees C) hatchling painted turtles, Chrysemys picta marginata. These genes encode (a) proteins involved in iron binding, (b) enzymes of antioxidant defense, and (c) serine protease inhibitors. The same genes were up-regulated by anoxia exposure (4 h of N2 gas exposure at 5 degrees C) of the hatchlings which suggests that these defenses for freeze tolerance are aimed at counteracting the injurious effects of the ischemia imposed by plasma freezing.     

Strategies for exploration of freeze responsive gene expression: advances in vertebrate freeze tolerance

Winter survival for many cold-blooded species involves freeze tolerance, the capacity to endure the freezing of a high percentage of total body water as extracellular ice. The wood frog (Rana sylvatica) is the primary model animal used for studies of vertebrate freeze tolerance and current studies in my lab are focused on the freeze-induced changes in gene expression that support freezing survival. Using cDNA library screening, we have documented the freeze-induced up-regulation of a number of genes in wood frogs including both identifiable genes (fibrinogen, ATP/ADP translocase, and mitochondrial inorganic phosphate carrier) and novel proteins (FR10, FR47, and Li16). All three novel proteins share in common the presence of hydrophobic regions that may indicate that they have an association with membranes, but apart from that each shows unique tissue distribution patterns, stimulation by different signal transduction pathways and responses to two of the component stresses of freezing, anoxia, and dehydration. The new application of cDNA array screening technology is opening up a whole new world of possibilities in the search for molecular mechanisms that underlie freezing survival. Array screening of hearts from control versus frozen frogs hints at the up-regulation of adenosine receptor signaling for the possible mediation of metabolic rate suppression, hypoxia inducible factor mediated adjustments of anaerobic metabolism, natriuretic peptide regulation of fluid dynamics, enhanced glucose transporter capacity for cryoprotectant accumulation, defenses against the accumulation of advanced glycation end products, and improved antioxidant defenses as novel parts of natural freeze tolerance that remain to be explored.     

Aquaporin expression correlates with freeze tolerance in baker's yeast,and overexpression improves freeze tolerance in industrial strains

Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freeze-resistant baker's yeast strains for use in frozen dough applications.     

Decomposing health: tolerance and resistance to parasites in animals

Plant biologists have long recognized that host defence against parasites and pathogens can be divided into two conceptually different components: the ability to limit parasite burden (resistance) and the ability to limit the harm caused by a given burden (tolerance). Together these two components determine how well a host is protected against the effects of parasitism. This distinction is useful because it recognizes that hosts that are best at controlling parasite burdens are not necessarily the healthiest. Moreover, resistance and tolerance can be expected to have different effects on the epidemiology of infectious diseases and host-parasite coevolution. However, studies of defence in animals have to date focused on resistance, whereas the possibility of tolerance and its implications have been largely overlooked. The aim of our review is to (i) describe the statistical framework for analysis of tolerance developed in plant science and how this can be applied to animals, (ii) review evidence of genetic and environmental variation for tolerance in animals, and studies indicating which mechanisms could contribute to this variation, and (iii) outline avenues for future research on this topic.     

Unusual organic osmolytes in deep-sea animals: adaptations to hydrostatic pressure and other perturbants

Shallow-living marine invertebrates use free amino acids as cellular osmolytes, while most teleosts use almost no organic osmolytes. Recently we found unusual osmolyte compositions in deep-sea animals. Trimethylamine N-oxide (TMAO) increases with depth in muscles of some teleosts, skates, and crustaceans (up to 300 mmol/kg at 2900 m). Other deep-sea animals had high levels of (1). scyllo-inositol in echinoderms, gastropods, and polychaetes, (2). that polyol plus beta-alanine and betaine in octopods, (3). hypotaurine, N-methyltaurine, and unidentified methylamines in vestimentiferans from hydrothermal vents and cold seeps, and (4). a depth-correlated serine-phosphate osmolyte in vesicomyid clams from trench seeps. We hypothesize that some of these solutes counteract effects of hydrostatic pressure. With lactate dehydrogenase, actin, and pyruvate kinase, 250 mM TMAO (but not glycine) protected both ligand binding and protein stability against pressure. To test TMAO in living cells, we grew yeast under pressure. After 1 h at 71 MPa, 3.5 h at 71 MPa, and 17 h at 30 MPa, 150 mM TMAO generally doubled the number of cells that formed colonies. Sulfur-based osmolytes which are not correlated with depth, such as hypotaurine and thiotaurine, are probably involved in sulfide metabolism and detoxification. Thus deep-sea osmolytes may have at least two other roles beyond acting as simple compatible osmotica.     

Aquaporin expression and freeze tolerance in Candida albicans

Aquaporins are members of the major intrinsic protein superfamily of integral membrane proteins which enable the transport of water, glycerol, and other solutes across membranes in various organisms. In microorganisms, the physiological role of aquaporins is not yet defined. We found a clear correlation between expression of the Candida albicans aquaporin-encoding gene AQY1 and freeze tolerance. A connection with the function for the aquaporin in the natural environment of C. albicans is, however, not obvious.     

Cold tolerance of Littorinidae from southern Africa: intertidal snails are not constrained to freeze tolerance

All intertidal gastropods for which cold tolerance strategies have been assessed have been shown to be freeze tolerant. Thus, freeze tolerance is considered an adaptation to the intertidal environment. We investigated the cold tolerance strategies of three species of subtropical and temperate snails (Gastropoda: Littorinidae) to determine whether this group is phylogenetically constrained to freeze tolerance. We exposed dry acclimated and wet rehydrated snails to low temperatures to determine temperature of crystallisation (Tc), lower lethal temperature and LT₅₀ and to examine the relationship between ice formation and mortality. Tc was lowest in dry Afrolittorina knysnaensis (–13.6±0.4 °C), followed by dry Echinolittorina natalensis (–10.9±0.2 °C) and wet A. knysnaensis (–10.2±0.2 °C). The Tc of both A. knysnaensis and E. natalensis increased with rehydration, whereas Tc of dry and wet Afrolittorina africana did not differ (–9.6±0.2 and –9.0±0.2 °C respectively). Wet snails of all species exhibited no or low survival of inoculative freezing, whereas dry individuals of A. knysnaensis could survive subzero temperatures above –8 °C when freezing was inoculated with ice. In the absence of external ice, Afrolittorina knysnaensis employs a freeze-avoidance strategy of cold tolerance, the first time this has been reported for an intertidal snail, indicating that there is no family-level phylogenetic constraint to freeze tolerance. Echinolittorina natalensis and A. africana both showed pre-freeze mortality and survival of some internal ice formation, but were not cold hardy in any strict sense.     

Cellular and molecular adaptations to injurious mechanical stress

Transient disruptions of plasma membrane integrity--'wounds'--are frequently suffered by cells of gut, skin, muscle and the aorta, organs that are normally subjected to mechanical stress in vivo.As a protection against such potentially fatal mechanically induced injuries, cells may employ specialized submembranous proteins that mechanically reinforce the plasma membrane and thus prevent wounding or, should wounding occur, they may assemble a cytoskeletal structure to aid wound healing. Membrane wounds may provide a route out of the cytoplasm for basic fibroblast growth factor, explaining how a growth factor that lacks a conventional signal peptide sequence can act extracellularly.     

Climate variations and the physiological basis of temperature dependent biogeography: systemic to molecular hierarchy of thermal tolerance in animals

The physiological mechanisms limiting and adjusting cold and heat tolerance have regained interest in the light of global warming and associated shifts in the geographical distribution of ectothermic animals. Recent comparative studies, largely carried out on marine ectotherms, indicate that the processes and limits of thermal tolerance are linked with the adjustment of aerobic scope and capacity of the whole animal as a crucial step in thermal adaptation on top of parallel adjustments at the molecular or membrane level. In accordance with Shelford's law of tolerance decreasing whole animal aerobic scope characterises the onset of thermal limitation at low and high pejus thresholds (pejus=getting worse). The drop in aerobic scope of an animal indicated by falling oxygen levels in the body fluids and or the progressively limited capacity of circulatory and ventilatory mechanisms. At high temperatures, excessive oxygen demand causes insufficient oxygen levels in the body fluids, whereas at low temperatures the aerobic capacity of mitochondria may become limiting for ventilation and circulation. Further cooling or warming beyond these limits leads to low or high critical threshold temperatures (T(c)) where aerobic scope disappears and transition to an anaerobic mode of mitochondrial metabolism and progressive insufficiency of cellular energy levels occurs. The adjustments of mitochondrial densities and their functional properties appear as a critical process in defining and shifting thermal tolerance windows. The finding of an oxygen limited thermal tolerance owing to loss of aerobic scope is in line with Taylor's and Weibel's concept of symmorphosis, which implies that excess capacity of any component of the oxygen delivery system is avoided. The present study suggests that the capacity of oxygen delivery is set to a level just sufficient to meet maximum oxygen demand between the average highs and lows of environmental temperatures. At more extreme temperatures only time limited passive survival is supported by anaerobic metabolism or the protection of molecular functions by heat shock proteins and antioxidative defence. As a corollary, the first line of thermal sensitivity is due to capacity limitations at a high level of organisational complexity, i.e. the integrated function of the oxygen delivery system, before individual, molecular or membrane functions become disturbed. These interpretations are in line with the more general consideration that, as a result of the high level of complexity of metazoan organisms compared with simple eukaryotes and then prokaryotes, thermal tolerance is reduced in metazoans. A similar sequence of sensitivities prevails within the metazoan organism, with the highest sensitivity at the organismic level and wider tolerance windows at lower levels of complexity. However, the situation is different in that loss in aerobic scope and progressive hypoxia at the organismic level define the onset of thermal limitation which then transfers to lower hierarchical levels and causes cellular and molecular disturbances. Oxygen limitation contributes to oxidative stress and, finally, denaturation or malfunction of molecular repair, e.g. during suspension of protein synthesis. The sequence of thermal tolerance limits turns into a hierarchy, ranging from systemic to cellular to molecular levels.     

Oxygen supply and the adaptations of animals in groundwater

1. The first part of this review focuses on the oxygen status of natural groundwater systems (mainly porous aquifers) and hyporheic zones of streams. The second part examines the sensitivity of groundwater organisms, especially crustaceans, to low oxygen concentrations (< 3.0 mg L^?1 O₂). 2. Dissolved oxygen (DO) in groundwater is spatially heterogeneous at macro- (km), meso- (m) and micro- (cm) scales. This heterogeneity, an essential feature of the groundwater environment, reflects changes in sediment composition and structure, groundwater flow velocity, organic matter content, and the abundance and activity of micro-organisms. Dissolved oxygen also exhibits strong temporal changes in the hyporheic zone of streams as well as in the recharge area of aquifers, but these fluctuations should be strongly attenuated with increasing distance from the stream and the recharge zone. 3. Dissolved oxygen gradients along flow paths in groundwater systems and hyporheic zones vary over several orders of magnitude (e.g. declines of 9 × 10^?5 to 1.5 ×10^?2 mg L^?1 O₂ m^?1 in confined aquifers and 2 × 10^?2 to 1 mg L^?1 O₂ m^?1 in parafluvial water). Several factors explain this strong variation. Where the water table is close to the surface, oxygen is likely to be consumed rapidly in the first few metres below the water table because of incomplete degradation of soil-generated labile dissolved organic carbon (DOC) in the vadose zone. Where the water table is far from the surface, strong oxygen depletion in the vicinity of the water table does not occur, DO being then gradually consumed as groundwater flows down the hydraulic gradient. In unconfined groundwater systems, oxygen consumption along flow paths may be compensated by down-gradient replenishment of DO, resulting either from the ingress of atmospheric oxygen or water recharge through the vadose zone. In confined groundwater systems, where replenishment of oxygen is impossible, the removal time of DO varies from a few years to more than 10 000 years, depending mainly on the organic carbon content of the sediment. Comparison of the hyporheic zones between systems also revealed strong differences in the removal time and length of underground pathways for DO. This strong variability among systems seems related to differences in contact time of water with sediment. 4. Although groundwater macro-crustaceans are much more resistant to hypoxia than epigean species, they cannot survive severe hypoxia (DO < 0.01 mg L^?1 O₂) for very long (lethal time for 50% of the population ranged from 46.7 to 61.7 h). In severe hypoxia, none of the hypogean crustaceans examined utilized a high-ATP yielding metabolic pathway. High survival times are mainly a result of the combination of three mechanisms: a high storage of fermentable fuels (glycogen and phosphagen), a low metabolic rate in normoxia, and a further reduction in metabolic rate by reducing locomotion and ventilation. It is suggested here that the low metabolic rate of many hypogean species may be an adaptation to low oxygen and not necessarily result from an impoverished food supply. 5. An interesting physiological feature of hypogean crustaceans is their ability to recover from anaerobic stress and, more specifically, rapidly to resynthesize glycogen stores during post-hypoxic recovery. A high storage and rapid restoration of fermentable fuels (without feeding) allows groundwater crustaceans to exploit a moving mosaic of suboxic (< 0.3 mg L?1 O₂), dysoxic (0.3–3.0 mg L^?1 O₂) and oxic (> 3 mg L^?1 O₂) patches. 6. It is concluded that although hypogean animals are probably unsuited for life in extensively or permanently suboxic groundwater, they can be found in small or temporarily suboxic patches. Indeed, their adaptations to hypoxia are clearly suited for life in groundwater characterized by spatially heterogeneous or highly dynamic DO concentrations. Their capacity to survive severe hypoxia for a few days and to recover rapidly would explain partly why ecological field studies often reveal the occurrence of interstitial taxa in groundwater with a wide range of DO.     

Development, molecular composition and freeze tolerance of bovine embryos cultured in TCM-199 supplemented with hyaluronan

Hyaluronan (HA) is glycosaminoglycan that is present from the start of embryonic development and its role and concentration increases with embryo development. The objective of this study was to evaluate if the presence of HA in TCM-199 culture medium had an effect on the development and quality of bovine embryos. There was no effect of HA on the total number of zygotes developing to blastocysts on day 7, however more expanded and hatched blastocyst stages were observed on days 8 and 9 in the group supplemented with HA (p<0.05). Following freeze/thawing, significantly more (p<0.05) embryos cultured in medium supplemented with HA hatched than those cultured in TCM-199 alone or those with BSA. Medium supplemented with HA and BSA significantly increased the level of expression of glucose metabolism Glut-1 gene and embryo compaction Cx43 gene (p<0.05), and had no effect on Glut-5 and IGF-II expression. In addition, HA presence in culture decreased the level of expression of apoptosis Bax and oxidative stress SOX genes (p<0.05). There was significant difference in total number of nuclei between TCM-199 medium only and the remaining media containing BSA or HA plus BSA, between which there was no difference. In summary, our results indicate that the addition of high molecular weight HA to TCM-199 medium that contains BSA on day 4 of culture improved embryo development to hatching and hatched blastocysts and the quality of produced embryos, which were superior to embryos cultured without HA addition.     

Physiological and molecular adaptations to drought in Andean potato genotypes

The drought stress tolerance of two Solanum tuberosum subsp. andigena landraces, one hybrid (adgxtbr) and Atlantic (S. tuberosum subsp. tuberosum) has been evaluated. Photosynthesis in the Andigena landraces during prolonged drought was maintained significantly longer than in the Tuberosum (Atlantic) line. Among the Andigena landraces, 'Sullu' (SUL) was more drought resistant than 'Negra Ojosa' (NOJ). Microarray analysis and metabolite data from leaf samples taken at the point of maximum stress suggested higher mitochondrial metabolic activity in SUL than in NOJ. A greater induction of chloroplast-localized antioxidant and chaperone genes in SUL compared with NOJ was evident. ABA-responsive TFs were more induced in NOJ compared with SUL, including WRKY1, mediating a response in SA signalling that may give rise to increased ROS. NOJ may be experiencing higher ROS levels than SUL. Metabolite profiles of NOJ were characterized by compounds indicative of stress, for example, proline, trehalose, and GABA, which accumulated to a higher degree than in SUL. The differences between the Andigena lines were not explained by protective roles of compatible solutes; hexoses and complex sugars were similar in both landraces. Instead, lower levels of ROS accumulation, greater mitochondrial activity and active chloroplast defences contributed to a lower stress load in SUL than in NOJ during drought.     

Cyanobacterial metallothioneins: Biochemistry and molecular genetics

Summary Metallothioneins have been extensively studied in many different eukaryotes where they sequester, and hence detoxify, excess amounts of certain metal ions. However, the precise functions of many of these molecules are not fully understood. This article reviews literature concerning their namesakes in prokaryotes.Abbreviations MT metallothionein - MRE metal regulatory element     

Antioxidant status and genotypic tolerance of Ribes in vitro cultures to cryopreservation

Cold tolerance studies are required to improve understanding of how plant tissues survive and regenerate from cryogenic temperatures. Ribes genotypes with different survival responses following cryopreservation were examined to determine the role of oxidative stress and ethylene in cryo-injury. In vitro shoot cultures of Ribes ciliatum (cryo-sensitive) and Ribes nigrum (cryo-tolerant) were analysed for antioxidant status, hydroxyl radicals and ethylene production at different stages of an encapsulation-dehydration cryopreservation protocol. Differential genotypic responses occurred during sucrose-simulated cold acclimation, where tolerance was associated with greater increases in hydroxyl radical activity, antioxidant status, phenolic accumulation, anthocyanin pigmentation, and protein SH group status. Elevated antioxidant levels persisted through recovery in the more tolerant genotype, while no changes in oxidative stress markers were found in shoots recovered from the sensitive genotype. Genotypic differences in the production of the stress hormone ethylene also occurred during recovery, where the cryo-sensitive genotype produced more ethylene than the tolerant species. This study indicates elevated antioxidant status and phenolic accumulation may be determinants of cryogenic stress tolerance and that their manipulation could improve recovery after cryopreservation.     

Selected latest applications of molecular spectroscopy in natural product analysis

The field of molecular vibrational spectroscopy applied to natural products is advancing extremely fast. Traditionally applied separation techniques (LC-, μ-LC–MS, GC, CE-MS) offer the advantages of high selectivity/sensitivity, but their application for routine quality control is limited due to long analyses times. Therefore, molecular spectroscopy in combination with multivariate analysis (MVA) enjoys excellent reputation, because of the fast and non-invasive measurement enabling the analysis of several physical and chemical parameters simultaneously. Near infrared (NIR; 4.000–10.000 cm⁻¹), attenuated total reflection (ATR; 400–4.000 cm⁻¹), Raman and far ultraviolet (FUV;120–200 nm) spectroscopy have permanently increased their efficiencies for quality control of predominantly food stuff, but also of other natural products including mainly medicinal plants. All four techniques enable not only a quantitative analysis of potent ingredients, but also qualitative fingerprint analysis for the discrimination of, e.g., species and/or geographic origin, respectively. Thereby, each individual spectroscopic technique possesses its specific strength. Powerful miniaturized portable spectrometers based on linear variable tuneable filter (LVTF) or micro-electro-mechanical systems (MEMS) are helpful in order to prevent consumers from deception on one hand, on the other hand they represent powerful analytical instruments for measurements in the field. 2-Dimensional correlation spectroscopy (2DCOS) represents a powerful technique for monitoring the dynamics of a system including temperature stability, extraction procedures etc. Imaging and mapping spectroscopy using infrared radiation and/or Raman scattering are not only suitable for classification of food stuff including e.g. maize kernels and/or coffee beans, but also for localizing the distribution of ingredients down to a resolution of 4 μm.In the present contribution, the latest progresses of the different techniques are introduced and their applicability in the fields of natural product analysis will be discussed in detail by distinct selected applications.     

Differential molecular responses of rice and wheat coleoptiles to anoxia reveal novel metabolic adaptations in amino acid metabolism for tissue tolerance

Rice (Oryza sativa) and wheat (Triticum aestivum) are the most important starch crops in world agriculture. While both germinate with an anatomically similar coleoptile, this tissue defines the early anoxia tolerance of rice and the anoxia intolerance of wheat seedlings. We combined protein and metabolite profiling analysis to compare the differences in response to anoxia between the rice and wheat coleoptiles. Rice coleoptiles responded to anoxia dramatically, not only at the level of protein synthesis but also at the level of altered metabolite pools, while the wheat response to anoxia was slight in comparison. We found significant increases in the abundance of proteins in rice coleoptiles related to protein translation and antioxidant defense and an accumulation of a set of enzymes involved in serine, glycine, and alanine biosynthesis from glyceraldehyde-3-phosphate or pyruvate, which correlates with an observed accumulation of these amino acids in anoxic rice. We show a positive effect on wheat root anoxia tolerance by exogenous addition of these amino acids, indicating that their synthesis could be linked to rice anoxia tolerance. The potential role of amino acid biosynthesis contributing to anoxia tolerance in cells is discussed.     

Identification of chaperones in freeze tolerance in Saccharomyces cerevisiae

Exposure to low temperatures reduces protein folding rates and induces the cold denaturation of proteins. Considering the roles played by chaperones in facilitating protein folding and preventing protein aggregation, chaperones must exist that confer tolerance to cold stress. Here, yeast strains lacking individual chaperones were screened for reduced freezing tolerance. In total, 19 of 82 chaperone-deleted strains tested were more sensitive to freeze-thaw treatment than wild-type cells. The reintroduction of the respective chaperone genes into the deletion mutants recovered the freeze tolerance. The freeze sensitivity of the chaperone-knockout strains was also retained in the presence of 20% glycerol.