Frozen mountain pine needles: The endodermis discriminates between the ice-containing central tissue and the ice-free fully functional mesophyll.

Conifer (Pinaceae) needles are the most frost-hardy leaves. During needle freezing, the exceptional leaf anatomy, where an endodermis separates the mesophyll from the vascular tissue, could have consequences for ice management and photosynthesis. The eco-physiological importance of needle freezing behaviour was evaluated based on the measured natural freezing strain at the alpine treeline. Ice localisation and cellular responses to ice were investigated in mountain pine needles by cryo-microscopic techniques. Their consequences for photosynthetic activity were assessed by gas exchange measurements. The freezing response was related to the microchemistry of cell walls investigated by Raman microscopy. In frozen needles, ice was confined to the central vascular cylinder bordered by the endodermis. The endodermal cell walls were lignified. In the ice-free mesophyll, cells showed no freeze-dehydration and were found photosynthetically active. Mesophyll cells had lignified tangential cell walls, which adds rigidity. Ice barriers in mountain pine needles seem to be realised by a specific lignification patterning of cell walls. This, additionally, impedes freeze-dehydration of mesophyll cells and enables gas exchange of frozen needles. At the treeline, where freezing is a dominant environmental factor, the elaborate needle freezing pattern appears of ecological importance.

Freeze dehydration vs. supercooling of mesophyll cells: Impact of cell wall, cellular and tissue traits on the extent of water displacement.

The extent of freeze dehydration of mesophyll cells in response to extracellular ice varies from supercooling to severe freezing cytorrhysis. The structural factors involved are poorly understood. In a comparison of mesophyll cells of 11 species, the factors "cell wall", "cellular" and "tissue" traits were investigated. The extent of freeze dehydration was quantified as reduction in the sectional area during controlled freezing in the presence of ice. The cell wall thickness, cell size, cell area and the relative area of intercellular spaces were determined. The modulus of elasticity was determined by psychrometry. To grasp the relationships between factors and with freeze dehydration, we applied a principal component analysis. The first two components explain 84% of the variance in the dataset. The first principal component correlated negatively with the extent of freeze dehydration and relative area of intercellular spaces, and positively with the squared cell wall thickness to cell size ratio, elasticity and cell wall thickness. The cell size parameters determined the second principal component. Supercooling appeared preferable in cells with a high squared cell wall thickness to cell size ratio and a low relative area of intercellular spaces. Such factors are hypothesised to affect the magnitude of negative turgor pressure being built up below the turgor loss point. Negative turgor pressure slows dehydration by reducing the water potential gradient to the extracellular ice. With high levels of freeze dehydration, sufficient intercellular spaces for extracellular ice accommodation are needed. The low relative area of intercellular spaces increases cell-to-cell contact area and could support tissue stability.

Frozen in the dark: interplay of night-time activity of xanthophyll cycle, xylem attributes, and desiccation tolerance in fern resistance to winter.

While most ferns avoid freezing as they have a tropical distribution or shed their fronds, wintergreen species in temperate and boreoalpine ecosystems have to deal with sub-zero temperatures. Increasing evidence has revealed overlapping mechanisms of desiccation and freezing tolerance in angiosperms, but the physiological mechanisms behind freezing tolerance in ferns are far from clear. We evaluated photochemical and hydraulic parameters in five wintergreen fern species differing in their ability to tolerate desiccation. We assessed frond freezing tolerance, ice nucleation temperature and propagation pattern, and xylem anatomical traits. Dynamics of photochemical performance and xanthophyll cycle were evaluated during freeze-thaw events under controlled conditions and, in selected species, in the field. Only desiccation-tolerant species, which possessed a greater fraction of narrow tracheids (<18 µm) than sensitive species, tolerated freezing. Frond freezing occurred in the field at -3.4 ± 0.9 °C (SD) irrespective of freezing tolerance, freezable water content, or tracheid properties. Even in complete darkness, maximal photochemical efficiency of photosystem II was down-regulated concomitantly with zeaxanthin accumulation in response to freezing. This was reversible upon re-warming only in tolerant species. Our results suggest that adaptation for freezing tolerance is associated with desiccation tolerance through complementary xylem properties (which may prevent risk of irreversible cavitation) and effective photoprotection mechanisms. The latter includes de-epoxidation of xanthophylls in darkness, a process evidenced for the first time directly in the field.

Winter Nights during Summer Time: Stress Physiological Response to Ice and the Facilitation of Freezing Cytorrhysis by Elastic Cell Wall Components in the Leaves of a Nival Species.

Ranunculus glacialis grows and reproduces successfully, although the snow-free time period is short (2-3 months) and night frosts are frequent. At a nival site (3185 m a.s.l.), we disentangled the interplay between the atmospheric temperature, leaf temperatures, and leaf freezing frequency to assess the actual strain. For a comprehensive understanding, the freezing behavior from the whole plant to the leaf and cellular level and its physiological after-effects as well as cell wall chemistry were studied. The atmospheric temperatures did not mirror the leaf temperatures, which could be 9.3 °C lower. Leaf freezing occurred even when the air temperature was above 0 °C. Ice nucleation at on average -2.6 °C started usually independently in each leaf, as the shoot is deep-seated in unfrozen soil. All the mesophyll cells were subjected to freezing cytorrhysis. Huge ice masses formed in the intercellular spaces of the spongy parenchyma. After thawing, photosynthesis was unaffected regardless of whether ice had formed. The cell walls were pectin-rich and triglycerides occurred, particularly in the spongy parenchyma. At high elevations, atmospheric temperatures fail to predict plant freezing. Shoot burial prevents ice spreading, specific tissue architecture enables ice management, and the flexibility of cell walls allows recurrent freezing cytorrhysis. The peculiar patterning of triglycerides close to ice rewards further investigation.

Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants.

Low temperature stress has a severe impact on the distribution, physiology, and survival of plants in their natural habitats. While numerous studies have focused on the physiological and molecular adjustments to low temperatures, this study provides evidence that cold induced physiological responses coincide with distinct ultrastructural alterations. Three plants from different evolutionary levels and habitats were investigated: The freshwater alga Micrasterias denticulata, the aquatic plant Lemna sp., and the nival plant Ranunculus glacialis. Ultrastructural alterations during low temperature stress were determined by the employment of 2-D transmission electron microscopy and 3-D reconstructions from focused ion beam-scanning electron microscopic series. With decreasing temperatures, increasing numbers of organelle contacts and particularly the fusion of mitochondria to 3-dimensional networks were observed. We assume that the increase or at least maintenance of respiration during low temperature stress is likely to be based on these mitochondrial interconnections. Moreover, it is shown that autophagy and degeneration processes accompany freezing stress in Lemna and R. glacialis. This might be an essential mechanism to recycle damaged cytoplasmic constituents to maintain the cellular metabolism during freezing stress.

Deep supercooling enabled by surface impregnation with lipophilic substances explains the survival of overwintering buds at extreme freezing.

The frost survival mechanism of vegetative buds of angiosperms was suggested to be extracellular freezing causing dehydration, elevated osmotic potential to prevent freezing. However, extreme dehydration would be needed to avoid freezing at the temperatures down to -45°C encountered by many trees. Buds of Alnus alnobetula, in common with other frost hardy angiosperms, excrete a lipophilic substance, whose functional role remains unclear. Freezing of buds was studied by infrared thermography, psychrometry, and cryomicroscopy. Buds of A. alnobetula did not survive by extracellular ice tolerance but by deep supercooling, down to -45°C. An internal ice barrier prevented ice penetration from the frozen stem into the bud. Cryomicroscopy revealed a new freezing mechanism. Until now, supercooled buds lost water towards ice masses that form in the subtending stem and/or bud scales. In A. alnobetula, ice forms harmlessly inside the bud between the supercooled leaves. This would immediately trigger intracellular freezing and kill the supercooled bud in other species. In A. alnobetula, lipophilic substances (triterpenoids and flavonoid aglycones) impregnate the surface of bud leaves. These prevent extrinsic ice nucleation so allowing supercooling. This suggests a means to protect forestry and agricultural crops from extrinsic ice nucleation allowing transient supercooling during night frosts.

Solar irradiation levels during simulated long- and short-term heat waves significantly influence heat survival, pigment and ascorbate composition, and free radical scavenging activity in alpine Vaccinium gaultherioides.

In the 20th century, annual mean temperatures in the European Alps rose by almost 1 K and are predicted to rise further, increasing the impact of temperature on alpine plants. The role of light in the heat hardening of plants is still not fully understood. Here, the alpine dwarf shrub Vaccinium gaultherioides was exposed in situ to controlled short-term heat spells (150 min with leaf temperatures 43-49°C) and long-term heat waves (7 days, 30°C) under different irradiation intensities. Lethal leaf temperatures (LT50 ) were calculated. Low solar irradiation [max. 250 photosynthetic photon flux density (PPFD)] during short-term heat treatments mitigated the heat stress, shown by reduced leaf tissue damage and higher Fv /Fm (potential quantum efficiency of photosystem 2) than in darkness. The increase in xanthophyll cycle activity and ascorbate concentration was more pronounced under low light, and free radical scavenging activity increased independent of light conditions. During long-term heat wave exposure, heat tolerance increased from 3.7 to 6.5°C with decreasing mean solar irradiation intensity (585-115 PPFD). Long-term exposure to heat under low light enhanced heat hardening and increased photosynthetic pigment, dehydroascorbate and violaxanthin concentration. In conclusion, V. gaultherioides is able to withstand temperatures of around 50°C, and its heat hardening can be enhanced by low light during both short- and long-term heat treatment. Data showing the specific role of light during short- and long-term heat exposure and the potential risk of lethal damage in alpine shrubs as a result of rising temperature are discussed.

Complex bud architecture and cell-specific chemical patterns enable supercooling of Picea abies bud primordia.

Bud primordia of Picea abies, despite a frozen shoot, stay ice free down to -50 °C by a mechanism termed supercooling whose biophysical and biochemical requirements are poorly understood. Bud architecture was assessed by 3D-reconstruction, supercooling and freezing patterns by infrared video thermography, freeze dehydration and extraorgan freezing by water potential measurements, and cell-specific chemical patterns by Raman microscopy and mass spectrometry imaging. A bowl-like ice barrier tissue insulates primordia from entrance by intrinsic ice. Water repellent and densely packed bud scales prevent extrinsic ice penetration. At -18 °C, break-down of supercooling was triggered by intrinsic ice nucleators whereas the ice barrier remained active. Temperature-dependent freeze dehydration (-0.1 MPa K-1 ) caused accumulation of extraorgan ice masses that by rupture of the shoot, pith tissue are accommodated in large voids. The barrier tissue has exceptionally pectin-rich cell walls and intercellular spaces, and the cell lumina were lined or filled with proteins, especially near the primordium. Primordial cells close to the barrier accumulate di, tri and tetrasaccharides. Bud architecture efficiently prevents ice penetration, but ice nucleators become active inside the primordium below a temperature threshold. Biochemical patterns indicate a complex cellular interplay enabling supercooling and the necessity for cell-specific biochemical analysis.

Ultrasonic emissions during ice nucleation and propagation in plant xylem.

Ultrasonic acoustic emission analysis enables nondestructive monitoring of damage in dehydrating or freezing plant xylem. We studied acoustic emissions (AE) in freezing stems during ice nucleation and propagation, by combining acoustic and infrared thermography techniques and controlling the ice nucleation point. Ultrasonic activity in freezing samples of Picea abies showed two distinct phases: the first on ice nucleation and propagation (up to 50 AE s(-1) ; reversely proportional to the distance to ice nucleation point), and the second (up to 2.5 AE s(-1) ) after dissipation of the exothermal heat. Identical patterns were observed in other conifer and angiosperm species. The complex AE patterns are explained by the low water potential of ice at the ice-liquid interface, which induced numerous and strong signals. Ice propagation velocities were estimated via AE (during the first phase) and infrared thermography. Acoustic activity ceased before the second phase probably because the exothermal heating and the volume expansion of ice caused decreasing tensions. Results indicate cavitation events at the ice front leading to AE. Ultrasonic emission analysis enabled new insights into the complex process of xylem freezing and might be used to monitor ice propagation in natura.

Application of heat stress in situ demonstrates a protective role of irradiation on photosynthetic performance in alpine plants.

The impact of sublethal heat on photosynthetic performance, photosynthetic pigments and free radical scavenging activity was examined in three high mountain species, Rhododendron ferrugineum, Senecio incanus and Ranunculus glacialis using controlled in situ applications of heat stress, both in darkness and under natural solar irradiation. Heat treatments applied in the dark reversibly reduced photosynthetic performance and the maximum quantum efficiency of photosystem II (Fv /Fm), which remained impeded for several days when plants were exposed to natural light conditions subsequently to the heat treatment. In contrast, plants exposed to heat stress under natural irradiation were able to tolerate and recover from heat stress more readily. The critical temperature threshold for chlorophyll fluorescence was higher under illumination (Tc (')) than in the dark (Tc). Heat stress caused a significant de-epoxidation of the xanthophyll cycle pigments both in the light and in the dark conditions. Total free radical scavenging activity was highest when heat stress was applied in the dark. This study demonstrates that, in the European Alps, heat waves can temporarily have a negative impact on photosynthesis and, importantly, that results obtained from experiments performed in darkness and/or on detached plant material may not reliably predict the impact of heat stress under field conditions.

Is sexual reproduction of high-mountain plants endangered by heat?

Strong solar irradiation in combination with still air and dry soil can cause prostrate high-mountain plants to heat up considerably and ultimately suffer heat damage. Such heat damage has been repeatedly shown for vegetative structures, but not for reproductive structures, which we expected to be particularly vulnerable to heat. Heat effects on cold-adapted plants may increase with rising global temperatures and the predicted increase in heat waves. We have tested the heat tolerance of reproductive versus vegetative shoots at different reproductive stages, comparing ten common plant species from different elevation belts in the European Alps. Plant samples were exposed to temperatures in 2-K steps of 30 min each between 42 and 56 °C. Heat damage was assessed by visual rating and vital staining. Reproductive shoots were on average 2.5 K less heat tolerant (LT50, i.e. the mean temperature causing 50 % heat damage, 47.2 °C) than vegetative shoots (mean LT50 49.7 °C). Initial heat injuries (mean LT10) were observed at 43-45 °C in heat-susceptible species and at 45-47 °C in more heat-tolerant species, in at least one reproductive stage. Generally, heat tolerance was significantly higher during fruiting than during the bud stages and anthesis. Prostrate species with acaulescent buds and flowers tolerated heat better than those with caulescent buds and flowers. Petals were the most heat-susceptible plant structure and mature pollen the most heat tolerant. Based on these data, heat tolerance of reproductive structures appears to be adapted to the prevailing maximum temperatures which the plants experience during different reproductive stages in their environment. During hot spells, however, heat tolerance thresholds may be exceeded. More frequent heat waves would decrease the reproductive output and, consequently, the competitiveness of heat-susceptible species.

Ice barriers promote supercooling and prevent frost injury in reproductive buds, flowers and fruits of alpine dwarf shrubs throughout the summer.

Over-wintering reproductive buds of many woody plants survive frost by supercooling. The bud tissues are isolated from acropetally advancing ice by the presence of ice barriers that restrict ice growth. Plants living in alpine environments also face the risk of ice formation in summer months. Little knowledge exists, how reproductive structures of woody alpine plants are protected from frost injury during episodic summer frosts. In order to address this question, frost resistance of three common dwarf shrubs, Calluna vulgaris, Empetrum hermaphroditum and Loiseleuria procumbens was measured and ice formation and propagation were monitored in twigs bearing reproductive shoots during various stages of reproductive development (bud, anthesis, and fruit) throughout the alpine summer. Results indicated that, in the investigated species, ice barriers were present at all reproductive stages, isolating the reproductive shoots from ice advancing from the subtending vegetative shoot. Additionally, in the reproductive stems ice nucleating agents that are active at warm, sub-zero temperatures, were absent. The ice barriers were 100% effective, with the exception of L. procumbens, where in 13% of the total observations, the ice barrier failed. The ice barriers were localized at the base of the pedicel, at the anatomical junction of the vegetative and reproductive shoot. There, structural aspects of the tissue impede or prevent ice from advancing from the frozen stem into the pedicel of the reproductive shoot. Under the experimental conditions used in this study, ice nucleation initially occurred in the stem of the vegetative shoot at species-specific mean temperatures in the range of -4.7 to -5.8 °C. Reproductive shoots, however, remained supercooled and ice free down to a range of -7.2 to -18.2 °C or even below -22 °C, the lowest temperature applied in the study. This level of supercooling is sufficient to prevent freezing of reproductive structures at the lowest air temperature occurring at the altitude of the upper distribution boundary of the natural habitat of the investigated species which is between -8 and -10 °C in summer. Frost resistance assays indicated that reproductive shoots are much less frost resistant than vegetative stems, and in contrast to vegetative shoots, are not ice tolerant. Supercooling of reproductive shoots in alpine, woody plant species is an effective mechanism that protects developing offspring from potential frost damage resulting from episodic summer freezing events.

Frost resistance of reproductive tissues during various stages of development in high mountain plants.

Frost resistance of reproductive vs aboveground vegetative structures was determined for six common European high alpine plant species that can be exposed to frosts throughout their whole reproductive cycle. Freezing tests were carried out in the bud, anthesis and fruit stage. Stigma and style, ovary, placenta, ovule, flower stalk/peduncle and, in Ranunculus glacialis, the receptacle were separately investigated. In all species, the vegetative organs tolerated on an average 2-5 K lower freezing temperatures than the most frost-susceptible reproductive structures that differed in their frost resistance. In almost all species, stigma, style and the flower stalk/peduncle were the most frost-susceptible reproductive structures. Initial frost damage (LT10) to the most susceptible reproductive structure usually occurred between -2 and -4°C independent of the reproductive stage. The median LT50 across species for stigma and style ranged between -3.4 and -3.7°C and matched the mean ice nucleation temperature (-3.7 ± 1.4°C). In R. glacialis, the flower stalk was the most frost-susceptible structure (-5.4°C), and was in contrast to the other species ice-tolerant. The ovule and the placenta were usually the most frost-resistant structures. During reproductive development, frost resistance (LT50) of single reproductive structures mostly showed no significant change. However, significant increases or decreases were also observed (2.1 ± 1.2 K). Reproductive tissues of nival species generally tolerated lower temperatures than species occurring in the alpine zone. The low frost resistance of reproductive structures before, during and shortly after anthesis increases the probability of frost damage and thus, may restrict successful sexual plant reproduction with increasing altitude.

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.

Heat Acclimation under Drought Stress Induces Antioxidant Enzyme Activity in the Alpine Plant Primula minima.

Heat and drought stresses are increasingly relevant topics in the context of climate change, particularly in the Alps, which are warming faster than the global average. Previously, we have shown that alpine plants, including Primula minima, can be gradually heat hardened under field conditions in situ to achieve maximum tolerance within a week. Here, we investigated the antioxidant mechanisms of P. minima leaves that had been heat hardened (H) without or with (H+D) additional drought stress. Lower free-radical scavenging and ascorbate concentrations were found in H and H+D leaves, while concentrations of glutathione disulphide (GSSG) were higher under both treatments without any change in glutathione (GSH) and little change in glutathione reductase activity. In contrast, ascorbate peroxidase activity in H leaves was increased, and H+D leaves had >two-fold higher catalase, ascorbate peroxidase and glucose-6-phosphate dehydrogenase activities compared with the control. In addition, the glutathione reductase activity was higher in H+D compared with H leaves. Our results highlight that the stress load from heat acclimation to maximum tolerance is associated with a weakened low-molecular-weight antioxidant defence, which may be compensated for by an increased activity of antioxidant enzymes, particularly under drought conditions.

Seasonal Xylem Sap Acidification Is Governed by Tree Phenology, Temperature and Elevation of Growing Site.

pH of xylem sap (pHx) was determined in three trees (Malus domestica (apple tree), Picea abies and Pinus cembra) in response to seasonal changes. Conifer trees from lowland (600 m) were compared to trees growing at the alpine timberline (1950 m a.s.l.). Xylem sap was extracted with a Scholander pressure bomb and pHx was measured with a pH microsensor. In all species, pHx changed markedly with season. In spring, pHx was acidic; during winter, the pHx was more alkaline. In apple trees, the pHx did not show a significant correlation with temperature but was rather affected by developmental stage. During flushing in spring, xylem sap acidification took place concomitant to the developmental stage "tight cluster", when foliar development enables a significant transpiration and a consequent movement of water in the xylem. The xylem sap of the two studied conifers showed a significantly larger seasonal alkalinisation (+2.1) than found in apple trees (+1.2) and was significantly more pronounced at the timberline. Xylem sap acidification took place before bud break. pHx had a significant negative correlation with soil temperatures and corresponded to already reported pHx of angiosperms. Overall, pHx appears to be a sensitive stress marker and indicator of activity status in tree xylem.

Parallel Differentiation and Plastic Adjustment of Leaf Anatomy in Alpine Arabidopsis arenosa Ecotypes.

Functional and structural adjustments of plants in response to environmental factors, including those occurring in alpine habitats, can result in transient acclimation, plastic phenotypic adjustments and/or heritable adaptation. To unravel repeatedly selected traits with potential adaptive advantage, we studied parallel (ecotypic) and non-parallel (regional) differentiation in leaf traits in alpine and foothill ecotypes of Arabidopsis arenosa. Leaves of plants from eight alpine and eight foothill populations, representing three independent alpine colonization events in different mountain ranges, were investigated by microscopy techniques after reciprocal transplantation. Most traits clearly differed between the foothill and the alpine ecotype, with plastic adjustments to the local environment. In alpine populations, leaves were thicker, with altered proportions of palisade and spongy parenchyma, and had fewer trichomes, and chloroplasts contained large starch grains with less stacked grana thylakoids compared to foothill populations. Geographical origin had no impact on most traits except for trichome and stomatal density on abaxial leaf surfaces. The strong parallel, heritable ecotypic differentiation in various leaf traits and the absence of regional effects suggests that most of the observed leaf traits are adaptive. These trait shifts may reflect general trends in the adaptation of leaf anatomy associated with the colonization of alpine habitats.

Seed dormancy in alpine species.

In alpine species the classification of the various mechanisms underlying seed dormancy has been rather questionable and controversial. Thus, we investigated 28 alpine species to evaluate the prevailing types of dormancy. Embryo type and water impermeability of seed coats gave an indication of the potential seed dormancy class. To ascertain the actual dormancy class and level, we performed germination experiments comparing the behavior of seeds without storage, after cold-dry storage, after cold-wet storage, and scarification. We also tested the light requirement for germination in some species. Germination behavior was characterized using the final germination percentage and the mean germination time. Considering the effects of the pretreatments, a refined classification of the prevailing dormancy types was constructed based on the results of our pretreatments. Only two out of the 28 species that we evaluated had predominantly non-dormant seeds. Physiological dormancy was prevalent in 20 species, with deep physiological dormancy being the most abundant, followed by non-deep and intermediate physiological dormancy. Seeds of four species with underdeveloped embryos were assigned to the morphophysiologial dormancy class. An impermeable seed coat was identified in two species, with no additional physiological germination block. We defined these species as having physical dormancy. Light promoted the germination of seeds without storage in all but one species with physiological dormancy. In species with physical dormancy, light responses were of minor importance. We discuss our new classification in the context of former germination studies and draw implications for the timing of germination in the field.

Protective Role of Ice Barriers: How Reproductive Organs of Early Flowering and Mountain Plants Escape Frost Injuries.

In the temperate zone of Europe, plants flowering in early spring or at high elevation risk that their reproductive organs are harmed by episodic frosts. Focusing on flowers of two mountain and three early-flowering colline to montane distributed species, vulnerability to ice formation and ice management strategies using infrared video thermography were investigated. Three species had ice susceptible flowers and structural ice barriers, between the vegetative and reproductive organs, that prevent ice entrance from the frozen stems. Structural ice barriers as found in Anemona nemorosa and Muscari sp. have not yet been described for herbaceous species that of Jasminum nudiflorum corroborates findings for woody species. Flowers of Galanthus nivalis and Scilla forbesii were ice tolerant. For all herbs, it became clear that the soil acts as a thermal insulator for frost susceptible below ground organs and as a thermal barrier against the spread of ice between individual flowers and leaves. Both ice barrier types presumably promote that the reproductive organs can remain supercooled, and can at least for a certain time-period escape from effects of ice formation. Both effects of ice barriers appear significant in the habitat of the tested species, where episodic freezing events potentially curtail the reproductive success.