Metabolite profiling reveals novel multi-level cold responses in the diploid model Fragaria vesca (woodland strawberry)

Winter freezing damage is a crucial factor in overwintering crops such as the octoploid strawberry (Fragaria × ananassa Duch.) when grown in a perennial cultivation system. Our study aimed at assessing metabolic processes and regulatory mechanisms in the close-related diploid model woodland strawberry (Fragaria vesca L.) during a 10-days cold acclimation experiment. Based on gas chromatography/time-of-flight-mass spectrometry (GC/TOF-MS) metabolite profiling of three F. vesca genotypes, clear distinctions could be made between leaves and non-photosynthesizing roots, underscoring the evolvement of organ-dependent cold acclimation strategies. Carbohydrate and amino acid metabolism, photosynthetic acclimation, and antioxidant and detoxification systems (ascorbate pathway) were strongly affected. Metabolic changes in F. vesca included the strong modulation of central metabolism, and induction of osmotically-active sugars (fructose, glucose), amino acids (aspartic acid), and amines (putrescine). In contrast, a distinct impact on the amino acid proline, known to be cold-induced in other plant systems, was conspicuously absent. Levels of galactinol and raffinose, key metabolites of the cold-inducible raffinose pathway, were drastically enhanced in both leaves and roots throughout the cold acclimation period of 10 days. Furthermore, initial freezing tests and multifaceted GC/TOF-MS data processing (Venn diagrams, independent component analysis, hierarchical clustering) showed that changes in metabolite pools of cold-acclimated F. vesca were clearly influenced by genotype.     

Cold acclimation induces freezing tolerance via antioxidative enzymes, proline metabolism and gene expression changes in two chrysanthemum species

Cold acclimation is necessary for chrysanthemum to achieve its genetically determined maximum freezing tolerance, but the underlying physiological and molecular mechanisms are unclear. The aim of this study was to discover whether changes in antioxidative enzymes, proline metabolism and frost-related gene expression induced by cold acclimation are related to freezing tolerance. Our results showed that the semi-lethal temperature (LT₅₀) decreased from ?7.3 to ?23.5 °C in Chrysanthemum dichrum and ?2.1 to ?7.1 °C in Chrysanthemum makinoi, respectively, after cold acclimation for 21 days. The activities of SOD, CAT and APX showed a rapid and transient increase in the two chrysanthemum species after 1 day of cold acclimation, followed by a gradual increase during the subsequent days and then stabilization. qRT-PCR analysis showed that the expression levels of some isozyme genes (Mn SOD, CAT and APX) were upregulated, which was consistent with the SOD, CAT and APX activities, while others remained relatively constant (Fe SOD and Cu/Zn SOD). P5CS and PDH expression were increased under cold acclimation and the level of P5CS presented similar trends as proline content, indicating proline accumulation was via P5CS and PDH cooperation. Cold acclimation also promoted DREB, COR413 and CSD gene expression. The activities of three enzymes and gene expression were higher in C. dichrum than in C. makinoi after cold acclimation. Our data suggested that cold-inducible freezing-tolerance could be attributed to higher activity of antioxidant enzymes, and increased proline content and frost-related gene expression during different periods.     

Freezing tolerance, cold acclimation and oxidative stress in potato. Paraquat tolerance is related to acclimation but is a poor indicator of freezing tolerance

Potato is a species commonly cultivated in temperate areas where the growing season may be interrupted by frosts, resulting in loss of yield. Cultivated potato, Solanum tuberosum, is freezing sensitive, but it has several freezing-tolerant wild potato relatives, one of which is S. commersonii. Our study was aimed to resolve the relationship between enhanced freezing tolerance, acclimation capacity and capacity to tolerate active oxygen species. To be able to characterize freezing tolerant ideotypes, a potato population (S1), which segregates in freezing tolerance, acclimation capacity and capacity to tolerate superoxide radicals, was produced by selfing a somatic hybrid between a freezing-tolerant Solanum commersonii (LT₅₀=-4.6°C) and -sensitive S. tuberosum (LT₅₀=-3.0°C). The distribution of non-acclimated freezing tolerance (NA-freezing tolerance) of the S1 population varied between the parental lines and we were able to identify genotypes, having significantly high or low NA-freezing tolerance. When a population of 25 genotypes was tested both for NA-freezing and paraquat (PQ) tolerance, no correlation was found between these two traits (R = 0.02). However, the most NA-freezing tolerant genotypes were also among the most PQ tolerant plants. Simultaneously, one of the NA-freezing sensitive genotypes (2022) (LT₅₀=-3.0°C) was observed to be PQ tolerant. These conflicting results may reflect a significant, but not obligatory, role of superoxide scavenging mechanisms in the NA-freezing tolerance of S. commersonii. The freezing tolerance after cold acclimation (CA-freezing tolerance) and the acclimation capacity (AC) was measured after acclimation for 7 days at 4/2°C. Lack of correlation between NA-freezing tolerance and AC (R =-0.05) in the S1 population points to independent genetic control of NA-freezing tolerance and AC in Solanum sp. Increased freezing tolerance after cold acclimation was clearly related to PQ tolerance of all S1 genotypes, especially those having good acclimation capacity. The rapid loss of improved PQ tolerance under deacclimation conditions confirmed the close relationship between the process of cold acclimation and enhanced PQ tolerance. Here, we report an increased PQ tolerance in cold-acclimated plants compared to non-acclimated controls. However, we concluded that high PQ tolerance is not a good indicator of actual freezing tolerance and should not be used as a selectable marker for the identification of a freezing-tolerant genotype.     

Differences in heat tolerance plasticity between supratidal and intertidal snails indicate complex responses to microhabitat temperature variation

Tropical intertidal gastropods that experience extreme and highly variable daily temperatures have evolved significant and complex heat tolerance plasticity, comprising components that respond to different timescales of temperature variation. An earlier study showed different plasticity attributes in snails from differently-heated coastlines, suggesting lifelong irreversible responses that matched habitat thermal regimes. To determine whether heat tolerance plasticity varied at a finer, within-shore spatial scale, we compared the responses of supratidal (predominantly shade-dwelling) and intertidal (frequently solar-exposed) populations of the tropical thermophilic gastropod, Echinolittorina malaccana. Snails modified lethal temperature (LT₅₀) under warm or cool laboratory acclimation, with the overall variation in LT₅₀ being greater in the supratidal (56.0–58.0 °C) than in the intertidal population (57.1–58.1 °C). Similar maximum LT₅₀s expressed by the populations after warm acclimation suggest a capacity limitation under these temperature conditons. The different minimum LT₅₀s after cool acclimation corresponded with microhabitat temperature and field acclimatization of the snails. Different responses to the same laboratory acclimation treatment imply long-term (and possibly lifelong) thermal acclimatization, which could benefit sedentary organisms that are randomly recruited as larvae from a common thermally-stable aquatic environment to thermally-unpredictable intertidal microhabitats. These findings provide another example of thermal tolerance plasticity operating at microhabitat scales, suggesting the importance of considering microhabitat thermal responses when assessing broad-scale environmental change.     

Temperature-dependent development, diapause and cold tolerance of Gratiana graminea, a potential biological control agent of Solanum viarum in Florida, USA

The objectives of this study were to examine temperature-dependent development, diapause and cold tolerance of Gratiana graminea Klug (Chrysomelidae), a candidate biological control agent of tropical soda apple, Solanum viarum Dunal (Solanaceae). Immature development was examined at six constant temperatures ranging from 15°C to 30°C. Diapause induction was determined by exposing adults to either long or short photoperiods at 20°C and cold tolerance was assessed by exposing adults to 0°C. G. graminea completed development at temperatures ranging from 20°C to 30°C. Linear regression estimated a lower temperature threshold of 11.7°C and 312 degree-days were required to complete development. Diapause was induced when adults were exposed to short photoperiods (10:14 L:D h) at 20°C. The lethal times for diapausing adults of G. graminea at 0°C (LT₅₀?=?19?days, LT₉₀?=?41?days) were two times higher compared to Gratiana boliviana Spaeth, a biological control agent already established in south and central Florida, USA. The presence of diapause and the greater cold tolerance suggest that G. graminea may establish and perform better than G. boliviana in northern Florida.     

How endangered is sexual reproduction of high-mountain plants by summer frosts? Frost resistance, frequency of frost events and risk assessment

In temperate-zone mountains, summer frosts usually occur during unpredictable cold spells with snow-falls. Earlier studies have shown that vegetative aboveground organs of most high-mountain plants tolerate extracellular ice in the active state. However, little is known about the impact of frost on reproductive development and reproductive success. In common plant species from the European Alps (Cerastium uniflorum, Loiseleuria procumbens, Ranunculus glacialis, Rhododendron ferrugineum, Saxifraga bryoides, S. moschata, S. caesia), differing in growth form, altitudinal distribution and phenology, frost resistance of reproductive and vegetative shoots was assessed in different reproductive stages. Intact plants were exposed to simulated night frosts between ?2 and ?14 °C in temperature-controlled freezers. Nucleation temperatures, freezing damage and subsequent reproductive success (fruit and seed set, seed germination) were determined. During all reproductive stages, reproductive shoots were significantly less frost resistant than vegetative shoots (mean difference for LT₅₀ ?4.2 ± 2.7 K). In most species, reproductive shoots were ice tolerant before bolting and during fruiting (mean LT₅₀ ?7 and ?5.7 °C), but were ice sensitive during bolting and anthesis (mean LT₅₀ around ?4 °C). Only R. glacialis remained ice tolerant during all reproductive stages. Frost injury in reproductive shoots usually led to full fruit loss. Reproductive success of frost-treated but undamaged shoots did not differ significantly from control values. Assessing the frost damage risk on the basis of summer frost frequency and frost resistance shows that, in the alpine zone, low-statured species are rarely endangered as long as they are protected by snow. The situation is different in the subnival and nival zone, where frost-sensitive reproductive shoots may become frost damaged even when covered by snow. Unprotected individuals are at high risk of suffering from frost damage, particularly at higher elevations. It appears that ice tolerance in reproductive structures is an advantage but not an absolute precondition for colonizing high altitudes with frequent frost events.     

Evergreen alpine shrubs have high freezing resistance in spring,irrespective of snowmelt timing and exposure to frost: an investigation from the Snowy Mountains,Australia

Over winter, alpine plants are protected from low-temperature extremes by a blanket of snow. Climate change predictions indicate an overall reduction in snowpack and an earlier thaw; a situation which could expose the tips of shrubs which extend above the snowpack to freezing events in early spring, and cause foliar frost damage during the onset of physiological activity. We assessed the photosynthetic responses of freezing-damaged shrub leaves from an assay of freezing temperatures in the Snowy Mountains in south-eastern Australia, using chlorophyll fluorometery ex situ. We sampled leaves that were exposed early during the spring thaw and leaves that were buried in snow for up to two extra weeks, from four evergreen shrub species at monthly intervals following the period of snowmelt. Freezing resistance (estimated from LT₅₀) was poorest at the earliest spring sampling time, in both exposed above-snow and protected below-snow foliage in all species. Protected foliage in early spring had lower freezing resistance than exposed foliage, but not significantly so. By the third sampling time, freezing resistance was significantly better in the lower protected foliage (LT₅₀ of ? 14) compared with the upper exposed foliage (LT₅₀ of ? 10) in one species. Over the course of spring, freezing resistance improved significantly in all species, with LT₅₀ values of between ? 10 and ? 15 °C by the third sampling time, which is lower than the minimum air temperatures recorded at that time (> ? 5 °C). The results indicate that the dominant evergreen shrub species in this area may only be susceptible to freezing events very early in spring, before a period of frost-hardening occurs after snowmelt. Later in spring, these alpine shrubs appear frost hardy, thus further perpetuating the positive feedbacks surrounding shrub expansion in alpine areas.     

Survival of cabbage stem flea beetle larvae,Psylliodes chrysocephala,exposed to low temperatures

The cabbage stem flea beetle, Psylliodes chrysocephala (L.) (Coleoptera: Chrysomelidae), is a major pest of winter oilseed rape. The larvae live throughout winter in leaf petioles and stems. Winter temperatures might play an important role in survival during winter and hence population dynamics, yet to what degree is unknown. This study investigates the effect of exposure time, cold acclimation, and larval stage on survival at ?5 and ?10 °C. Exposure time at ?5 °C was 1, 2, 4, 8, 12, 16, and 20 days and 6, 12, 24, 36, 48, 72, 96, 120, and 144 h at ?10 °C. Mortality increased with increasing exposure time and was significantly lower for cold‐acclimated larvae. Estimated time until an expected mortality of 50% (LT₅₀) and 90% (LT₉₀) of larvae exposed to ?5 °C was 7.4 and 9.6 days (non‐acclimated) and 11.0 and 15.1 days (acclimated), respectively. Estimated LT₅₀ for non‐acclimated and acclimated larvae exposed to ?10 °C was 32.6 and 70.5 h, respectively, and estimated LT₉₀ 66.8 and 132.2 h. Significant differences in mortality between larval stages were observed only at ?5 °C. When exposed to ?5 °C for 8 days, mortality of first and second instars was 81.2 and 51.3%, respectively. When exposed to ?10 °C for 2 days, mortality of first and second instars was 70.5 and 76.1%. Data on winter temperatures in Denmark from 1990 to 2013 showed that larvae were rarely exposed to a number of continuous days at ?5 or ?10 °C causing a potential larval mortality of 50–90%.     

Reduced clonal reproduction indicates low potential for establishment of hybrids between wild and cultivated strawberries (Fragaria vesca × F. × ananassa)

The genus Fragaria (Rosaceae) contains 24 species, including hybrid species such as the garden strawberry (Fragaria × ananassa Duch.). Natural hybridization between Fragaria species has repeatedly been reported, and studies on the hybridization potential between F. × ananassa and its wild relatives have become increasingly important with the outlook for genetically modified garden strawberries. In Europe, a candidate species for hybridization with garden strawberries is the common woodland strawberry (Fragaria vesca L.). Although a previous field survey indicated that the potential for hybridization between F. vesca and F. × ananassa is low, it is not clear whether the lack of natural hybrids is caused by known pre- and postzygotic barriers, or whether hybrid plants lack the fitness to establish in natural F. vesca populations. We grew different F. vesca and F. vesca × F. × ananassa hybrid clones with and without competition in a greenhouse and assessed biomass production, clonal reproduction, and sexual reproduction of plants. While some hybrid clones exceeded F. vesca in biomass production, general clonal reproduction was much lower and delayed in hybrids. Furthermore, hybrids were sterile. These results demonstrate a mechanism by which the general lack of F. vesca × F. × ananassa hybrids in natural habitats can be explained, in addition to the known low hybridization potential between garden and woodland strawberries. We conclude that hybrids have a competitive disadvantage against co-occurring F. vesca plants due to inferior and delayed clonal reproduction, and that the potential for hybrid establishment under natural conditions is low.     

Olive (Olea europaea L.) freezing tolerance related to antioxidant enzymes activity during cold acclimation and non acclimation

The changes in the antioxidant enzymes activity, total protein and proline content and their correlations with freezing tolerance (FT) (expressed as LT₅₀) were investigated at 11 different olive cultivars at cold-acclimation (CA, in February) and non-acclimation (NA, in August) stages. Leaf samples were collected from each cultivar and were divided into two groups. The first group was immediately frozen in liquid nitrogen for further biochemical analysis. The second ones was subjected to different freezing temperatures (?5, ?10, ?15 and ?20 °C) for 10 h, in order to determine their FT. The unfrozen control samples were kept at 4 °C. The results showed that Fishomi, Mission and Shengeh were the most freezing tolerant among other cultivars. In contrast, Zard, Manzanilla and Amigdalolia were the most sensitive ones. The cold acclimation enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), polyphenol oxidase (PPO) and total protein content. However, proline content and phenylalanine ammonia-lyase (PAL) activity did not change or even decreased slightly at CA stage, compare to those samples at NA stage. It was found that LT₅₀ to be closely correlated to POD, CAT, and PPO activity at CA and NA stages. Overall, higher leaf POD, CAT, and PPO activity could be used as important selection criteria in screening tolerant olive cultivars for cold zone climatic.     

Genotypic variation in cold tolerance influences the yield of Miscanthus

When grown in Europe, Miscanthus genotypes often produce yields lower than their potential due to late emergence of shoots in the spring or to damage from late frosts when shoots emerge too early. Here, we investigate genotypic variation in the base temperature (T_b) for shoot emergence and in the lethal temperature for shoots (LT₅₀) in four Miscanthus genotypes. In all genotypes, lowering temperature increased the time to shoot emergence, with T_b ranging from 8.6°C in Sac‐5 to 6°C in Sin‐H9. Frost treatments below ?8°C resulted in a marked reduction in growth in all four genotypes. Sin‐H9 was the most frost tolerant with an LT₅₀ of ?9.3°C. There was little variation found in leaf osmotic potential, but leaf moisture content was significantly lower in Sin‐H9 than in the other genotypes. The lower thermal requirement for emergence and lower LT₅₀ seen in Sin‐H9 was incorporated into a model of Miscanthus production. The model showed an extended growing season that was predicted to increase yields by up to 25%.     

Cold-induced changes affect survival after exposure to vitrification solution during cryopreservation in the south-west Australian Mediterranean climate species Lomandra sonderi (Asparagaceae)

There is limited knowledge of the effects of exposure to low temperatures in the unique Mediterranean climate plant species of Western Australia. We have thus investigated the effect of low temperature on cryogenic tolerance in Lomandra sonderi, an endemic perennial species of southwest Western Australia. Lomandra sonderi plants were preconditioned in tissue culture at constant 23 °C (12 h light/dark cycle) or alternating 20/?1 °C (16 h light and 8 h dark cycle). Shoot tips from both conditions were analysed for their phospholipid, sterol and soluble sugar compositions. Shoot tips were also cryoexposed via a droplet-vitrification protocol. Survival in both preconditioning regimes for cryoexposed and non-cryoexposed samples was the same, but plants from the 20/?1 °C regime displayed an improved tolerance to the overall cryopreservation process in both cryoexposed and non-cryoexposed samples, thereby eliminating exposure to liquid nitrogen as a primary cause of reduced post-cryogenic viability. Preconditioning of in vitro shoots of L. sonderi at 20/?1 °C induced significant increases in phosphatidylcholine (from 7.30 ± 3.46 to 22.2 ± 7.80 ng mg^?1 FW) and increases in several soluble sugars (fructose, galactose, glucose, sucrose) compared to shoots incubated at 23 °C—changes consistent with known cold acclimation responses in plant species generally—but sterol content remained largely unchanged. Analysis of electrolyte leakage in shoot tips from both preconditioning regimes generated a significantly lower LT₅₀ value in the 20/?1 °C samples (?5.45 ± 0.53 °C) over the 23 °C samples (?2.5 ± 0.08 °C). Increased tolerance to cryoexposure in L. sonderi appears to lie mainly with acclimation-induced changes in membrane composition and promotion of membrane stability and hence increased resistance to freeze damage.     

Induced expression of the class II chitinase gene during cold acclimation and dehydration of bermudagrass (Cynodon sp.)

Bermudagrass cultivars vary greatly in their ability to survive freezing temperatures as a result of a differential ability to cold acclimate (CA) at temperatures slightly above 0°C. Little information exists on the genetic and physiological mechanisms associated with the cold acclimation process in bermudagrass. Experiments were conducted to study the changes in chitinase gene expression during cold acclimation of freeze-tolerant bermudagrass cultivars. A chitinase gene (CynCHT1) was isolated from ’Midiron’ bermudagrass. Because the hydrophilic protein putatively encoded by the gene lacked an N-terminal cysteine-rich domain and a hydrophobic C-terminal extension, it was classified a class II chitinase. The expression patterns of this and related chitinase genes in response to CA, drought, and ABA were investigated in freeze-tolerant ’MSU’ (LT₅₀=?11°C), Midiron (LT₅₀=?10°C) and ’Uganda’ (LT₅₀=?8°C) bermudagrasses. Northern-blot analysis indicated expression in the crown tissues induced by CA at 8°C/2°C day/night temperature cycles. Induction of gene expression was evident in tissues sampled at 2 and 28 days after initiating CA. Expression after 2-days de-acclimation at 28°C/24°C was similar to control levels. Significantly higher levels of CA-induced chitinase gene expression were observed in MSU and Midiron, compared to Uganda. Similar expression patterns were observed among the cultivars in responses to drought and ABA. These results suggest that chitinases have important roles in bermudagrass response to low temperature and dehydration stresses.     

Heat tolerance of early developmental stages of glacier foreland species in the growth chamber and in the field

In glacier forelands, seeds readily germinate, however, a high proportion of seedlings die shortly after their appearance. We hypothesized that besides drought, frost and missing safe sites, heat on the ground surface could be one of the major threats for seedlings. The heat strain in different ground strata was assessed from 2007 to 2010. The heat tolerance (LT₅₀) of eleven alpine species from different successional stages was tested considering imbibed (G1) and germinated seeds (G2) as well as seedlings (G3). Additionally, the heat hardening capacity of seedlings was determined in the field. Across all species, LT₅₀ decreased significantly by 9 K from G1 (55 °C) to G3 (46 °C), similarly in all species of the successional stages. Field-grown seedlings had mostly an increased LT₅₀ (2K). Intraspecifically, LT₅₀ of seedlings varied between 40.6 and 52.5 °C. Along the chronosequence, LT₅₀ in G1 was similar, but was higher in G2 and G3 of early successional species. The highest temperatures occurred at 0–0.5 cm in air (mean/absolute maximum: 42.6/54.1 °C) posing a significant heat injury risk for seedlings when under water shortage transpirational cooling is prevented. Below small stones (0–0.5 cm), maxima were 4 K lower, indicating heat safer microsites. Maxima >30 °C occurred at 32.3, >40 °C at 6.2 %. Interannually, 2010 was the hottest year with heat exceeding LT₅₀ at all microsites (0–0.5 cm). Temperature maxima on sandy surfaces were lower than on microsites with gravel (diameter <5–10 mm). The hot summer of 2010 may be a small foretaste of in future more severe and frequent heat waves. Ground surface temperature maxima at the pioneer stage are already now critical for heat survival and may partly explain the high seedling mortality recognized on recently deglaciated terrain.     

Temperature and salinity tolerances of larval Californian grunion, Leuresthes tenuis (Ayres): A comparison with gulf grunion, L. Sardina (Jenkins & Evermann)

Temperature and salinity tolerances were determined for larval California grunion, Leuresthes tenuis (Ayres), and compared with previous data for Gulf of California grunion, L. sardina (Jenkins & Evermann). Larvae of similar age and acclimation history showed little interspecific difference in thermal tolerance, as measured by half-hour LT₅₀ values for 20–30 day old late postlarvae acclimated at various temperatures, and by upper and lower incipient lethal temperatures for 18°C-acclimated prolarvae. The upper incipient lethal temperature differed by 1 deg.-C (32°C for L. tenuis, 31°C for L. sardina), while the lower incipient lethal temperature for the 18°C acclimated prolarvae of both species was 7.5°C. L. tenuis larvae were much less euryhaline than L. sardina, with incipient lethal salinities of 4.2–41 %. for prolarvae and 8.6–38 %. for 20-day-old postlarvae; comparable values for L. sardina are 4–67.5 %. and 5–57.5 %. Both species show a decrease in temperature and salinity tolerance with age. The larvae of these disjunct congeners show a significant physiological divergence in euryhalinity but not in overall temperature tolerance. These tolerances are discussed in relation to the respective geographic ranges and behavioral responses of the two species.     

Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection

Key message

We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics.

Abstract

Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12–16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1–2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying.     

Freezing cytorrhysis and critical temperature thresholds for photosystem II in the peat moss Sphagnum capillifolium

Leaflets of Sphagnum capillifolium were exposed to temperatures from ?5°C to +60°C under controlled conditions while mounted on a microscope stage. The resultant cytological response to these temperature treatments was successfully monitored using a light and fluorescence microscope. In addition to the observable cytological changes during freezing cytorrhysis and heat exposure on the leaflets, the concomitant critical temperature thresholds for inactivation of photosystem II (PS II) were studied using a micro fibre optic and a chlorophyll fluorometer mounted to the microscope stage. Chlorophyllous cells of S. capillifolium showed extended freezing cytorrhysis immediately after ice nucleation at ?1.1°C in the water in which the leaflets were submersed during the measurement. The occurrence of freezing cytorrhysis, which was visually manifested by cell shrinkage, was highly dynamic and was completed within 2 s. A total reduction of the mean projected diameter of the chloroplast containing area during freezing cytorrhysis from 8.9 to 3.8 μm indicates a cell volume reduction of approximately ?82%. Simultaneous measurement of chlorophyll fluorescence of PS II was possible even through the frozen water in which the leaf samples were submersed. Freezing cytorrhysis was accompanied by a sudden rise of basic chlorophyll fluorescence. The critical freezing temperature threshold of PS II was identical to the ice nucleation temperature (?1.1°C). This is significantly above the temperature threshold at which frost damage to S. capillifolium leaflets occurs (?16.1°C; LT₅₀) which is higher than observed in most higher plants from the European Alps during summer. High temperature thresholds of PS II were 44.5°C which is significantly below the heat tolerance of chlorophyllous cells (49.9°C; LT₅₀). It is demonstrated that light and fluorescence microscopic techniques combined with simultaneous chlorophyll fluorescence measurements may act as a useful tool to study heat, low temperature, and ice-encasement effects on the cellular structure and primary photosynthetic processes of intact leaf tissues.     

Hot or not? Comparative behavioral thermoregulation,critical temperature regimes,and thermal tolerances of the invasive lionfish <Emphasis Type="Italic">Pterois</Emphasis> sp. versus native western North Atlantic reef fishes

Temperature influences the geographic range, physiology, and behavior of many ectothermic species, including the invasive lionfish Pterois sp. Thermal parameters were experimentally determined for wild-caught lionfish at different acclimation temperatures (13, 20, 25 and 32 °C). Preferences and avoidance were evaluated using a videographic shuttlebox system, while critical thermal methodology evaluated tolerance. The lionfish thermal niche was compared experimentally to two co-occurring reef fishes (graysby Cephalopholis cruentata and schoolmaster Lutjanus apodus) also acclimated to 25 °C. The physiologically optimal temperature for lionfish is likely 28.7 ± 1 °C. Lionfish behavioral thermoregulation was generally linked to acclimation history; tolerance and avoidance increased significantly at higher acclimation temperatures, but final preference did not. The tolerance polygon of lionfish shows a strong correlation between thermal limits and acclimation temperature, with the highest CT_max at 39.5 °C and the lowest CT_min at 9.5 °C. The tolerance range of invasive lionfish (24.61 °C) is narrower than those of native graysby (25.25 °C) and schoolmaster (26.87 °C), mostly because of lower thermal maxima in the former. Results show that lionfish display “acquired” thermal tolerance at higher and lower acclimation temperatures, but are no more eurythermal than other tropical fishes. Collectively, these results suggest that while lionfish range expansion in the western Atlantic is likely over the next century from rising winter sea temperatures due to climate change, the magnitude of poleward radiation of this invasive species is limited and will likely be equivalent to native tropical and subtropical fishes with similar thermal minima.     

Freezing Tolerance and Solute Changes in Contrasting Genotypes of Lolium perenne L. Acclimated to Cold and Drought

Clonal ramets of 12 contrasting genotypes of Lolium perenneL. were grown in sand or soil-based compost and maintained underwell-watered conditions at 20/15°C or acclimated to lowtemperature (2°C) or to a restricted water supply. Freezingtolerance was measured as LT₅₀ following exposure to sub-zerotemperatures in a freezing tank. Measurements were also madeof osmotic potential, water-soluble carbohydrates, free proline,free amino acids, and minerals in entire tillers. Acclimationto both drought and cold lowered LT₅₀, induced osmotic adjustment,and increased concentrations of proline and amino acids, Rootingmedium had little effect on LT₅₀, but caused large differencesin osmotic potential and in proline and amino-acid concentrations.There was considerable genetic diversity for all charactersmeasured, except for mineral contents. There was, however, norelationship between LT₅₀ and osmotic potential or solute contentthat was consistent across the three sources of variation (growingmedium, acclimation, genotype). Furthermore, the diverse genotypicvalues of cold-induced freezing tolerance were not correlatedwith those of drought-induced tolerance. It is concluded thatmore precise measurements are needed of the partitioning ofsolutes during acclimation and of the sensitivity of differentorgans and tissues to freezing.Copyright 1993, 1999 AcademicPress Perennial ryegrass, hardening, acclimation, osmotic potential, solute potential, carbohydrates, proline