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.

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.

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.

Freezing pattern and frost killing temperature of apple (Malus domestica) wood under controlled conditions and in nature.

The freezing pattern and frost killing temperatures of apple (Malus domestica Borkh.) xylem were determined by differential thermal analysis and infrared differential thermal analysis (IDTA). Results from detached or attached twigs in controlled freezing experiments and during natural field freezing of trees were compared. Non-lethal freezing of apoplastic water in apple xylem as monitored during natural winter frosts in the field occurred at -1.9 ± 0.4 °C and did not change seasonally. The pattern of whole tree freezing was variable and specific to the environmental conditions. On detached twigs high-temperature freezing exotherms (HTEs) occurred 2.8 K below the temperature observed under natural frosts in the field with a seasonal mean of -4.7 ± 0.5 °C. Microporous apple xylem showed freezing without a specific pattern within a few seconds in IDTA images during HTEs, which is in contrast to macroporous xylem where a 2D freezing pattern mirrors anatomical structures. The pith tissue always remained unfrozen. Increasing twig length increased ice nucleation temperature; for increased twig diameter the effect was not significant. In attached twigs frozen in field portable freezing chambers, HTEs were recorded at a similar mean temperature (-4.6 ± 1.0 °C) to those for detached twigs. Upon lethal intracellular freezing of apple xylem parenchyma cells (XPCs) low-temperature freezing exotherms (LTEs) can be recorded. Low-temperature freezing exotherms determined on detached twigs varied significantly between a winter minimum of -36.9 °C and a summer maximum -12.7 °C. Within the temperature range wherein LTEs were recorded by IDTA in summer (-12.7 ± 0.5 to -20.3 ± 1.1 °C) various tiny clearly separated discontinuous freezing events could be detected similar to that in other species with contrasting XPC anatomy. These freezing events appeared to be initially located in the primary and only later in the secondary xylem. During the LTE no freezing events in the bark and central pith tissue were recorded. Attached twigs were exposed to various freezing temperatures at which LTEs occur. Even if 60% of XPCs were frost-damaged twigs were able to recuperate and showed full re-growth indicating a high regeneration capacity even after severe frost damage to XPCs.

Inflorescences of alpine cushion plants freeze autonomously and may survive subzero temperatures by supercooling.

Freezing patterns in the high alpine cushion plants Saxifraga bryoides, Saxifraga caesia, Saxifraga moschata and Silene acaulis were studied by infrared thermography at three reproductive stages (bud, anthesis, fruit development). The single reproductive shoots of a cushion froze independently in all four species at every reproductive stage. Ice formation caused lethal damage to the respective inflorescence. After ice nucleation, which occurred mainly in the stalk or the base of the reproductive shoot, ice propagated throughout that entire shoot, but not into neighboring shoots. However, anatomical ice barriers within cushions were not detected. The naturally occurring temperature gradient within the cushion appeared to interrupt ice propagation thermally. Consequently, every reproductive shoot needed an autonomous ice nucleation event to initiate freezing. Ice nucleation was not only influenced by minimum temperatures but also by the duration of exposure. At moderate subzero exposure temperatures (-4.3 to -7.7 °C) the number of frozen inflorescences increased exponentially. Due to efficient supercooling, single reproductive shoots remained unfrozen down to -17.4 °C (cooling rate 6 K h⁻¹). Hence, the observed freezing pattern may be advantageous for frost survival of individual inflorescences and reproductive success of high alpine cushion plants, when during episodic summer frosts damage can be avoided by supercooling.

Mesophyll freezing and effects of freeze dehydration visualized by simultaneous measurement of IDTA and differential imaging chlorophyll fluorescence.

Infrared differential thermal analysis (IDTA) and differential imaging chlorophyll fluorescence (DIF) were employed simultaneously to study the two-dimensional pattern of ice propagation in leaves and mesophyll freeze dehydration as detected by a significant increase of basic chlorophyll fluorescence (F(0)). IDTA and DIF technique gave different insights into the freezing process of leaves that was highly species-specific. IDTA clearly visualized the freezing process consisting of an initial fast spread of ice throughout the vascular system followed by mesophyll freezing. While mesophyll freezing was homogeneously in Poa alpina, Rhododendron ferrugineum and Senecio incanus as determined by IDTA, DIF showed a distinct pattern only in S. incanus, with the leaf tips being affected earlier. In Cinnamomum camphora, a mottled freezing pattern of small mesophyll compartments was observed by both methods. In IDTA images, a random pattern predominated, while in DIF images, compartments closer to lower order veins were affected earlier. The increase of F(0) following mesophyll freezing started after a species-specific time lag of up to 26 min. The start of the F(0) increase and its slope were significantly enhanced at lower temperatures, which suggest a higher strain on mesophyll protoplasts when freezing occurs at lower temperatures.

Ice propagation in plants visualized at the tissue level by infrared differential thermal analysis (IDTA).

We studied ice propagation in stems and leaves of various angiosperm deciduous and evergreen trees and shrubs and gymnosperms by differential thermal analysis (DTA) of thermal images captured with a digital infrared camera. This so-called infrared DTA (IDTA) technique should elucidate the smallest freezing exotherms in plant tissues. After intrinsic ice nucleation in the stem, ice spread into the leaves. On the leaf lamina, it was possible to differentiate between initial freezing in the veins and subsequent freezing in the mesophyll. The spread of ice followed the venation and could usually be tracked to third-order lateral veins. The exception was in mature leaves of Buxus sempervirens L., where large ice lenses formed in the central mesophyll lacunas. Longitudinal ice propagation within veins was faster (0.3-4.7 cm s(-1)) than measured in earlier studies (0.25-2.7 cm s(-1)). Peculiar leaf freezing patterns indicated that lateral ice propagation in the vascular bundles may take significantly longer than longitudinal ice propagation. Within the vascular bundle, the exact sites of initial vein freezing could not be resolved. However, the observed freezing patterns combined with chemical theory and anatomical structures suggest that initial freezing most probably occurs within the xylem vessels and tracheids. Ice barriers appeared to be present in leaf laminas of Cinnamomum camphora (L.) J. Presl., resulting in freezing of small mesophyll compartments produced by sclerenchymatic bundle sheath extensions. The IDTA was demonstrated to be a highly efficient method for resolving peculiarities of ice propagation at the plant tissue level.