# Monitoring of freezing dynamics in trees: a simple phase shift causes complexity

Guillaume Charrier, Markus Nolf, Georg Leitinger, Katline Charra-Vaskou, Adriano Losso, Ulrike Tappeiner, Thierry Ameglio, Stefan Mayr

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## To cite this version:

Guillaume Charrier, Markus Nolf, Georg Leitinger, Katline Charra-Vaskou, Adriano Losso, et al. Monitoring of freezing dynamics in trees: a simple phase shift causes complexity. Plant Physiology, 2017, 173 (4), pp.2196-2207. 10.1104/pp.16.01815. hal-01605135

HAL Id: hal-01605135

https://hal.science/hal-01605135v1

Submitted on 11 Dec 2024

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1 Short title: Monitoring of freezing dynamics in trees

2

3 Correspondence to: Dr. Guillaume Charrier

4 e-mail: guillaume.charrier@inra.fr

5 Phone: +33 5 40 00 36 64

6 Address: UMR 1202 Biodiversité Gènes & Communautés INRA/Université Bordeaux,

7 Bâtiment B2 - allée G. St Hilaire, CS 50023, 33615 Pessac Cedex - France

8

9

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11 Monitoring of freezing dynamics in trees: a simple phase shift causes complexity.

12 Authors

13 Charrier Guillaume<sup>1,2,3*</sup>, Nolf Markus<sup>1,4*</sup>, Leitinger Georg<sup>1</sup>, Charra-Vaskou Katline<sup>5</sup>, Losso

14 Adriano<sup>1</sup>, Tappeiner Ulrike<sup>1,6</sup>, Améglio Thierry<sup>5</sup>, Mayr Stefan<sup>1</sup>

15 <sup>1</sup> Department of Botany and Department of Ecology, University of Innsbruck, Sternwartestr.

16 15, A-6020 Innsbruck, Austria.

17 <sup>2</sup> Bordeaux Science Agro, Institut des Sciences de la Vigne et du Vin, Ecophysiologie et

18 Génomique Fonctionnelle de la Vigne, UMR 1287, F-33140 Villenave d'Ornon, France

19 <sup>3</sup> BIOGECO, INRA, Univ. Bordeaux, 33610 Cestas, France

20 <sup>4</sup> Hawkesbury Institute for the Environment, University of Western Sydney, Richmond, NSW

21 2753, Australia

22 <sup>5</sup> Université Clermont Auvergne, INRA, PIAF, F-6300 Clermont-Ferrand, France

23 <sup>6</sup> Institute for Alpine Environment, European Academy Bozen (EURAC), Drususallee 1,
24 39100 Bozen/Bolzano, Italy.

25 * Both authors contributed equally to this work

26 Authors' contributions: G.C., S.M., G.L. and M.N, and conceived the research plans; G.C.,

27 S.M. and M.N and performed the data monitoring (thermocouples, ultrasonic emissions and

28 dendrometers); G.L. performed the infrared imaging; A.L. conducted additional laboratory

29 experiments; G.C., M.N, G.L. and S.M. analyzed the data and wrote the article with

30 contributions of all the authors.

31 One-sentence summary

32 Monitoring of freezing in timberline trees via non-destructive methods revealed complex

33 spatial and temporal freezing patterns which promote internal water shifts and cavitation

34 events.
49 **Abstract**

50 During winter, trees have to cope with harsh conditions, including extreme freeze-thaw stress.

51 The present study focused on ice nucleation and propagation, related water shifts and xylem

52 cavitation as well as cell damage, and was based on *in situ* monitoring of xylem

53 (thermocouples) and surface temperatures (infrared imaging), ultrasonic emissions and

54 dendrometer analysis. Field experiments during late winter on *Picea abies* growing at the

55 alpine timberline revealed three distinct freezing patterns: (i) from the top of the tree towards

56 the base, (ii) from thin branches towards the main stem's top and base, and (iii) from the base

57 towards the top. Infrared imaging showed freezing within branches from their base towards

58 distal parts. Such complex freezing causes dynamic and heterogeneous patterns in water

59 potential and probably in cavitation. This study highlights the interaction between

60 environmental conditions upon freezing and thawing and demonstrates the enormous

61 complexity of freezing processes in trees. Diameter shrinkage, which indicated water fluxes

62 within the stem, and acoustic emission analysis, which indicated cavitation events near the ice

63 front upon freezing, were both related to minimum temperature, and upon thawing, related to

64 vapor pressure deficit and soil temperature. These complex patterns, emphasizing the

65 common mechanisms between frost and drought stress, shed new lights on understanding

66 winter tree physiology.

67 **Keywords:**

68 cavitation, dendrometer, freezing stress, ice propagation, infrared imaging, non-destructive

69 monitoring, ultrasonic acoustic emissions, xylem embolism.
70 **Introduction**

Plants in temperate biomes can contain water under its three physical states. Liquid water flows within the xylem according to the cohesion-tension theory (Dixon, 1896), and turns into vapor in leaves. During frost days, liquid water, at least partially, changes to ice. Alpine plants are an impressive example of plants coping with these phase shifts on a frequent basis, especially in late winter to early spring, when freeze-thaw cycles can occur daily (Mayr *et al.* 2006a).

Ice formation within plants influences their physiology mechanically, hydraulically and at a cellular level (Cinotti, 1991; Charrier *et al.*, 2013a; 2015a). Mechanical strain occurs as water expands during freezing (Kubler, 1983; Cinotti, 1991) and tension is induced in the remaining liquid-phase sap (Hansen & Beck, 1988; Charrier *et al.*, 2014a; Charra-Vaskou *et al.*, 2016).
Xylem cavitation is initiated on freezing due to the low (i.e. negative) water potential of ice (Charrier *et al.*, 2014a; Charra-Vaskou *et al.*, 2016) and the low solubility of gases in ice (Robson *et al.*, 1988; Sperry & Robson, 2001; Sevanto *et al.*, 2012). Embolism subsequently develops on thawing when gas bubbles released from the ice may coalesce, expand, and embolize the complete conduit (Sperry & Sullivan, 1992; Lemoine *et al.*, 1999; Pittermann & Sperry, 2003; Ball *et al.*, 2006; Mayr *et al.*, 2007; Mayr & Sperry, 2010; Sevanto *et al.*, 2012; Charrier *et al.*, 2014a; Charra-Vaskou *et al.*, 2016). The low water potential of ice also generates an osmotic disturbance on macro-molecule arrangement, which can lead to membrane breakage and cellular lysis (Pearce, 2001; Uemura *et al.*, 2006; Ruelland *et al.*, 2009).

When temperatures drop below 0°C, the xylem sap remains in a metastable, supercooled state (Wolfe & Bryant, 2001) until crystallization is initiated around a *nucleus*. Ice nucleation can occur when water molecules form long enough transitory hydrogen bonds with each other,
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reaching a critical nucleus size of 1.13 nm, containing *ca.* 190 molecules (*i.e.* homogeneous
nucleation at temperatures below -38°C), or onto a nucleating agent (heterogeneous
nucleation onto impurity, cell wall, or Ice Nucleation Active bacteria; Sakai & Larcher, 1987).

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In trees, ice nucleation is generally observed in the apoplastic compartment (Asahina, 1956),
through intrinsic heterogeneous nucleation (Wisniewski et al., 1997; Pearce, 2001). Once
started, ice propagates through the plant, radially and longitudinally (Neuner et al., 2010).

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The physical transition from a disordered liquid state to an ordered crystal structure is an
exothermic reaction, which releases latent heat (334 J·g⁻¹; Dereudre & Gazeau, 1992) and
entraps gas bubbles emerging from solution (Sperry & Robson, 2001; Sevanto et al., 2012). A
freezing exotherm can therefore be detected using thermocouples (e.g. Mayr et al., 2006a;
Pramsohler et al., 2012) or infrared imaging (Wisniewski et al., 1997; Hacker & Neuner,
2007; Charrier et al., 2015a). Exotherm monitoring is widely used to detect freezing in plants,
but this technique is very limited at detecting much slower phenomena such as thawing. At
thawing, xylem sap becomes liquid through an opposite endothermic reaction.

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Further insights into freezing and thawing processes were enabled by dendrometers (Zweifel
& Häsler, 2000) and ultrasonic emission analysis (Charrier *et al.*, 2015a). Dendrometers are
commonly used to monitor growth and drought stress in woody plants (Reineke, 1932;
Daubenmire, 1945; Deslauriers *et al.*, 2007; de Swaef *et al.*, 2015). However, upon freezing, a
sharp decrease in diameter is observed in the bark of acclimated plants, which is probably
caused by water fluxes towards the ice nucleation point in intercellular spaces (Zweifel &
Häsler, 2000; Ball *et al.*, 2006; Charra-Vaskou *et al.*, 2016). On thawing, the initial diameter
is restored, but only partly in plants that are not fully acclimated (Améglio *et al.*, 2001). The
proportion of irreversible diameter shrinkage can therefore serve as an assessment of frost-
induced damages to living cells (Lintunen *et al.*, 2015). Ultrasonic emission (UE; Mayr *et al.*,
2007) analysis is a non-destructive technique which detects spontaneous liquid to vapor phase

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transitions (i.e. cavitation events) under drought (Milburn, 1966; Tyree & Dixon, 1983; Mayr & Rosner, 2011; Vergeynst et al., 2014; Nolf et al., 2015) or frost stress (Kikuta & Richter 2003; Mayr et al., 2007; Charrier et al., 2014a). The latter is likely based on bubble formation due to the low water potential of the xylem sap near growing ice fronts. When the water potential reaches the cavitation threshold, gas bubbles form, and the sudden relaxation of water columns generates UEs. Recently, the UE technique was used to monitor ice propagation within branches in laboratory experiments (Charrier et al., 2015a), and UE velocity and attenuation were shown to enable the detection of ice in woody stems (Charrier et al. 2014b). UE were also observed during thawing, in relation with embolism formation according to the thaw-expansion hypothesis (Mayr & Sperry, 2010).

In trees, the dynamics of freezing and resulting occurrence of ice in the axis system have a crucial effect for hydraulics (e.g. increased levels of frost-induced embolism in distal parts of branches; Lemoine et al., 1999). Frozen xylem in the trunk blocks water uptake from the soil and thus supply to distal parts such as the leaves of evergreens. Ice in branches disconnects the hydraulic continuum within the plant and thus prevents equilibration of water potentials, which may cause complex within-plant variation in drought stress intensities (Mayr & Charra-Vaskou, 2007), especially when high evaporative demand and frozen soil are concomitant (Mayr et al., 2002; 2006b; 2014). This complexity is further increased by the spatio-temporal pattern of ice and respective frost damages in dead (xylem embolism) and living tissues (cell damage).

Ice nucleation usually occurs where the local (organ) temperature reaches the freezing point. Under field conditions, air temperature is often considered as a proxy for organ temperature. However, temperature varies spatially, being lower close to the ground in an open environment (Leuning & Cremer, 1988; Jordan & Smith, 1994; Battany, 2012) or, in more closed stands, at the air-plant interface (Tappeiner 1985; Winkel et al., 2009). Compared to
144 air temperature, plant organ temperature exhibits a buffered response due to thermal inertia of
145 the local wood section (proportional to stem diameter) and isolating layers (e.g. bark). The
146 actual temperature in plant tissues therefore is the result of the energy balance driven by net
147 radiation, air temperature, humidity and soil calorific capacity (Cellier, 1984; 1993; Leuning,
148 1988; Jordan & Smith, 1994). Thus, freezing and thawing patterns are complicated by
149 temperature variation, e.g. due to microclimatic factors, topography and crown architecture,
150 or the latent heat released by ice formation. Additionally, tissues with more negative osmotic
151 potentials exhibit lower freezing points (Sakai & Larcher, 1987; Yelenosky & Guy, 1989),
152 which in turn influences the spatial pattern of potential ice nucleation.

153 We hypothesized that late winter environmental conditions result in disturbances in the plant
154 hydraulic system, which generate ice formation and propagation from the coldest organs of
155 the tree (e.g. the apex, or thin side branches) towards the base. To test this hypothesis, we
156 monitored ice formation and propagation within the trunk and crown of *Picea abies* trees
157 growing near the alpine timberline using non-destructive methods (thermocouples, infrared
158 imaging, and UE analysis; Fig. 1). It is known that timberline trees are exposed to numerous
159 and intense freeze-thaw cycles (see above) and thus provide an ideal system to study
160 respective dynamics. Furthermore, an analysis of these patterns is essential to understand the
161 impact of winter conditions on tree physiology at higher elevation. Xylem (via
162 thermocouples) and surface temperature (via infrared imaging) were used to detect freezing
163 occurrence and ice propagation. UE analysis should enable to detect xylem sap cavitation,
164 either upon freezing or thawing, and dendrometry to detect water shifts and cellular damage,
165 in relation with environmental conditions.

166 **Results**
During four successive late winter periods, we recorded 15 independent freezing processes within the tree crown via exotherm detection in the xylem (thermocouples). Three different patterns of freezing exotherms within the crown, with respect to temporal and spatial courses, were observed: (i) from the top downwards to the basal part of the tree (approx. 20% of observations, Fig. 2A-B, March 19, 2013), (ii) from the base upwards to the tree top (approx. 30% of observations, Fig. 2C-D, March 22, 2013), (iii) from side branches to the stem and upwards (approx. 50% of observations, Fig. 2E-F, March 13, 2015). Ice propagated at approx. 1 to 4 mm·s⁻¹ in up- and downward directions.

Infrared image analysis allowed us to map changes in surface temperature across a large part of the tree, highlighting a more complex freezing pattern than indicated by exotherm analysis based on single, individually placed thermocouples. For instance on March 14, 2015 (Fig. 3; supplementary video 1), before freezing occurred (17:00), a vertical gradient in surface temperature was observed along the trunk from the middle (T₃, approx. -2°C) to the apex (T₁, approx. -2.5°C), and side branches (B approx. -3°C). From 17:46 to 18:15, exotherms indicated that freezing started from the branch base and moved towards the distal parts of branches, respectively. However, the pattern within the crown appeared as spatially erratic, with seemingly independent exotherms detected in different side branches. Overall, branches inserted at middle height froze before higher and lower branches. Although the increase in trunk surface temperature was too low to be visually detected on the video, temperature dynamics extracted from infrared image analysis (Fig. S1A, B) were in agreement with the dynamics observed via thermocouples (Fig. S1C, D). On side branches, exotherms were recorded earlier with thermocouples than with infrared imaging, because the latter recorded temperature changes at the surface while thermocouples were installed directly in the xylem. In the apical and middle parts of the trunk (T₁-T₃), exotherms were concurrently detected with thermocouple and infrared imaging, indicating a stronger exotherm signal based on a much
192 larger volume of freezing wood. The freezing patterns of a second tree (tree 2, at the right in 193 the supplementary video), and during other freezing events (March 25-27, 2014 or March 13-194 14, 2015) were similar, with side branches at middle height freezing before branches near the 195 apex or the base.

196 Upon freezing, UEs were detected concurrently with exotherm formation within the trunk as 197 measured by thermocouples (March 7, 2012; Fig. 4): from the lower ($T_5$, UE: 17:48; TC: 198 17:53) to the upper position ($T_2$, UE: 18:00; TC: 17:57). While the onset of UEs thus clearly 199 corresponded to freezing, consecutive acoustic activity did not exhibit a clear spatial pattern, 200 and UEs were detected long after the exotherm had dissipated (Fig. 5). Ultrasonic emissions 201 exhibited two phases of activity within a daily freeze-thaw cycle *i.e.* a phase from the onset of 202 freezing (indicated by a freezing exotherm) until sunrise, and a second phase after the xylem 203 had thawed in the morning until sunset (March 12-13, 2015, Fig. 5). Of the high number of 204 UEs recorded during the different monitoring periods (approx. 760,000 UE), 52% were 205 recorded in the side branch (B), 29% in the middle of the trunk ($T_3$), 15% in the upper portion 206 of the trunk (T2), and only 3.5% in the lower part (T5), with similar trends for day and 207 nighttime activities.

208 The two phases of acoustic activity were remarkably associated with decreasing trunk 209 diameter, however, to a different extent (March 12-13, 2015, Fig. 5). Daytime UE activity 210 (approx. 0.07 UEs·s⁻¹) and decreasing trunk diameter (-100µm; -0.01µm·s⁻¹) were observed as 211 long as temperature remained above 0°C (16:00). After sunset, the second phase of UE 212 activity (0.45 UEs·s⁻¹) started once the freezing exotherm reached a plateau (19:30), 213 concurrent with diameter shrinkage (-175µm; -0.12µm·s⁻¹). Finally, another peak in UE 214 activity (2.25 UEs·s⁻¹), was recorded when the xylem differential temperature (xylem 215 temperature - air temperature; $dT_{xylem-air}$ = +2.7°C at 0:00) started to decrease, again 216 associated with trunk diameter shrinkage (-400 µm; -0.12µm·s⁻¹; Fig. 5). During the
217 intermediate periods of low UE activity (e.g. plants thawing from 08:20 until 10:20, or at the
218 onset of freezing from 17:00 until 19:30) the stem recovered its initial diameter.

219 During March 12-17, 2015, we observed a period of pronounced daily temperature
220 fluctuations. Temperatures exhibited high diurnal amplitudes of 10-15K as well as increasing
221 minimum and maximum temperatures and corresponding vapor pressure deficits (Fig. 6A).
222 Since March 14, minimum temperature progressively increased, resulting in lower UE activity
223 and higher minimum diameter at night (Fig. 6B). However, UE activity during daytime
224 increased over this period, in relation with temperature (>5°C), and VPD (> 500 kPa).
225 Interestingly, nighttime freezing generated approx. 10 times higher diameter shrinkage than
226 daytime thawing, whereas maximum UE activity was three times lower at night than during
227 the day (Fig. 6B). Furthermore, UEs were almost completely restricted to periods of diameter
228 shrinkage (approx. 87% of total recorded UEs).

229 Nighttime UE activity and diameter shrinkage were negatively correlated with minimum air
230 temperature (R² = 0.509, P < 0.001 and R² = 0.410, P = 0.042 for UEs and diameter,
231 respectively; Fig. 7A; Tab. I). During the day, UE activity and diameter shrinkage were
232 correlated with minimum soil temperature (R² = 0.303, P < 0.001 and R² = 0.559, P = 0.008
233 for UEs and diameter, respectively; Fig. 7B). However, day- and nighttime UE activities were
234 related to temperatures via exponential functions, whereas diameter shrinkages via linear
235 functions. UE activity was, consequently, exponentially correlated to diameter shrinkage (R²
236 = 0.233, P = 0.031; Fig. 7C), with no significant difference between day and night (P =
237 0.124). Correlations between maximum VPD and UEs (P = 0.049), and between diameter
238 shrinkage and minimum air temperature were also observed (P = 0.002). Furthermore, the
239 maximum VPD observed during the same night was correlated with both nighttime UEs (P =
240 0.015) and diameter shrinkage (P < 0.001).
241 **Discussion**

242 This study shows that freezing plant tissues do not just undergo a simple phase shift. Instead,
243 UEs and stem diameter variation revealed that ice nucleation is only the onset of a complex
244 series of internal changes that continue several hours beyond the initial exotherm formation
245 (Fig. 5, Fig. 8). These changes may promote sap cavitation upon freezing and thawing in
246 environmental conditions typical for early spring (i.e. low air and soil temperature, high
247 VPD). Remarkably, even once most of the sap was in solid state, the frozen branch underwent
248 changes as indicated by subsequent radial stem (i.e. bark) shrinkage and increasing UE
249 activity with decreasing air temperatures (Fig. 6, Fig. 7, Tab. 1). Both of these effects were
250 correlated with soil temperature and VPD and likely resulted from the low water potential of
251 ice, which depends on the degree of supercooling (-1.16 MPa·K⁻¹; Hansen & Beck, 1988).
252 Upon freezing, radial water fluxes and related cavitation events increased the heterogeneity in
253 water distribution within the tree crown. Such conditions are expected to favor embolism
254 development on thawing, when the root system cannot compensate for evaporative water
255 losses (Fig. 8).

256 The remarkable correlation between diameter shrinkage and UE activity (Fig. 5-7) indeed
257 indicates that water fluxes are generated towards the ice nucleation point (Cavender-Bares,
258 2005; Ball et al., 2006). These water fluxes resulted in bark shrinkage and increased the
259 tension in the remaining liquid xylem sap (Charra-Vaskou et al., 2016). When the cavitation
260 threshold was reached, UE were generated (Charrier et al., 2014b), in relation with freezing
261 velocity (Charrier et al., 2015a). Furthermore, cavitation-related UEs may be generated when
262 air is expelled from the crystal lattice during freezing and gets pulled through pits (Mayr et
263 al., 2007; Charrier et al., 2014b; Charrier et al., 2015a). Such air bubbles can then expand at
264 thawing and, especially while the xylem sap is under tension (Mayr & Sperry, 2010), fill and
265 therefore embolize the whole conduit (Ball et al., 2006; Charra-Vaskou et al., 2016).
While ice nucleation and its related effects typically occur at night, UE activity and decreasing stem diameter were also observed during the day period (Fig. 5, Fig. 6, Tab. 1). Daytime UE activity increased with vapor pressure deficit (VPD) and decreasing soil temperature. The observed diameter shrinkage indicates that the water supply could not compensate the evaporative demand, resulting in increasing drought stress during sunny days ('winter drought'; Sakai & Larcher, 1987; Tranquillini, 1979), as previously described for mountainous areas (Michaelis, 1934a; 1934b; Henson, 1952; Larcher, 1957; 1977; Wardle, 1971; Tranquillini, 1979). Cavitation events thus occurred not only on freezing but also on thawing, which over the course of many freeze-thaw cycles can lead to high levels of winter embolism (e.g. Mayr et al., 2003; 2006b). Day- and nighttime environmental conditions therefore interact in water redistribution within the tree crown, which exponentially increases the probability of cavitation events (Fig. 7). Embolism repair and hydraulic recovery, which do not produce UEs, may mitigate this hydraulic limitation, and have indeed been observed in *Picea abies* even while the soil was frozen (Mayr et al., 2006b; 2014). Furthermore, insulation from VPD due to snow cover helps small trees and shrub species to avoid or reduce drought stress during winter months (Larcher, 1963).

Freeze-thaw cycles in winter and early spring are common in temperate to boreal and mountainous regions. The study site at the timberline and the late-winter season were chosen as the ideal place and time to observe them *in situ*, with broad daily thermal amplitudes due to topography, high elevation (1600 m a.s.l.), south-east exposure, and an open stand (Blennow, 1998; Blennow & Persson, 1998). Such conditions are particularly favorable for radiative freezing events to occur in late winter (Monteith & Unsworth, 1990; Lindkvist et al., 2000; Charrier et al., 2015b). During this period, when the sky is clear, the energy balance is usually positive at daytime (solar radiation > 600 W·m⁻²) and negative at night (long-wave infrared radiation dissipated to clear sky; Nobel, 1987; Snyder & Melo-Abreu, 2005). Air temperature
during the study period thus alternated on average from -4.4°C (nighttime) to +7°C (daytime), but extreme daily thermal amplitudes can reach up to 30 K in alpine conditions (Mayr et al., 2006a). Local sunset at the study site was between 16:00 and 17:00 (surrounding mountains approx. 20° above horizon), resulting in a negative net energy income of the ecosystem. A rapid decrease in air temperature (6.3 K·h⁻¹ on average) therefore led to fast freezing events. In the morning, the temperature increased at an even faster rate (8.1 K·h⁻¹ on average) and thus temperature changes were overall faster than generally reported (approx. 5 K·h⁻¹; Levitt, 1980; Sakai & Larcher, 1987). During the monitored period, the fastest rates of temperature change were similar for cooling (-14.5 K·h⁻¹; Apr 7, 2015) and warming (+15.8 K·h⁻¹; March 7, 2012). Cooling, however, was far slower than rates at which intracellular freezing and thus cellular damage was reported (-48 K·h⁻¹; Levitt, 1958; Siminovitch et al., 1978). Accordingly, stem diameters fully recovered to initial values after thawing (Fig. 5, Fig. 6), indicating that living tissues were still intact (Améglio et al., 2001; Lintunen et al., 2015; Charra-Vaskou et al., 2016). Also minimum temperatures were not critical, as plants can usually suffer temperatures as low as -40°C without significant cell damage (Christersson, 1978; Repo, 1992; Charra-Vaskou et al., 2012) before de-acclimation during the spring period.

The delay between the onset of exotherm formation (17:00) and diameter shrinkage (20:35), as well as the two phases observed in diameter shrinkage (before/after 21:50) suggest that the spatiotemporal pattern of freezing was heterogeneous (Fig. 5). Although exotherms observed via thermocouples revealed freezing of xylem tissue, the diameter remained unaffected for one to three hours (Fig. 5, Fig. 6). As long as the stem remained warmer than air, the ice water potential did not compensate the intra-cellular osmotic potential (Cavender-Bares 2005; Charrier et al., 2013b), nor reach the cavitation threshold (Charrier et al., 2015b). However, as the exothermal heat dissipated, liquid water was pulled from the cytoplasm toward the apoplasm, leading to cell plasmolyis (Uemura et al., 2006; Ruelland et al., 2009), diameter
316 shrinkage (Charra Vaskou et al., 2016) and UEs (Kasuga et al., 2014; Charrier et al., 2015b).
317 Because of their higher elasticity, bark tissues exhibit more pronounced thickness changes
318 upon freezing than xylem (Zweifel & Hasler, 2000; Améglio et al., 2001). The extracellular
319 freezing of the bark tissues was probably the trigger of water fluxes outwards from the xylem,
320 and the resulting tension generated UE. Other elements than the xylem conduits have been
321 shown to generate UEs on freezing in angiosperms, such as bark (Kikuta, 2003) or xylem
322 parenchyma (Kasuga et al., 2014).
323 Ice nucleation is partly a stochastic process i.e. freezing depends on the interaction between
324 low temperature and the presence of a nucleus. In our study, the lowest temperatures were
325 generally observed in the apex and branches, whereas, surprisingly, freezing sometimes also
326 started from the base of the trunk (Fig. 2 A-F, 3). We observed three distinct freezing patterns
327 in the same *Picea abies* tree (Fig. 2, 8): (i) Ice propagated from the base, when this part
328 remained frozen during the entire day while the upper crown thawed in the sun. (ii) Ice
329 nucleation occurred in the apex and the ice then spread downwards. (iii) Freezing started in
330 branches, with the ice front seemingly moving towards the main axis. These last two freezing
331 patterns would suggest that the distal parts of the tree (apex or branches) experienced lower
332 temperatures, which therefore had increased probability for nucleation. However, infrared
333 image analysis showed a more detailed picture: ice nucleation in branches occurred close to
334 the base of the branch and propagated towards the distal ends (see supplementary video).
335 These observations do not only show that the presence of metallic sensors in contact with the
336 xylem did not induce ‘artificial’ ice nucleation. They also suggest that a lower temperature in
337 branches (compared to the trunk), combined with larger conduits (at the branch base
338 compared to distal branch sections) may increase the probability for ice nucleation (Asahina,
339 1956; Lintunen et al., 2013) and, in consequence, make the base of branches a likely ice
340 nucleation site. Additional freezing experiments in the laboratory, where the direction of ice
propagation was determined in excised branch segments, could not confirm this pattern consistently (data not shown). However, these laboratory experiments could not simulate the microclimatic effect of dense needle cover in the distal portion of the branch, which might have buffered the temperature decrease.

Once nucleated, the ice front can propagate after the exothermal heat has dissipated through the bark (Shibkov *et al.*, 2003), whereby propagation rates within the water column are proportional to the degree of supercooling (Langer *et al.*, 1978; Rauschenberger *et al.*, 2013). In excised branches from *Picea abies*, propagation rates similar to the present field study were observed (2 and 4 mm·s⁻¹, for 1.5 and 2 K of supercooling, respectively; Charrier *et al.*, 2015a). Ice propagation towards the distal end of a branch can generate two opposite effects on sap water potential: (i) The density of ice with respect to liquid water (917 vs 1000 kg·m⁻³ at 0°C, respectively) induces an approx. 9% increase in local pressure (Hare & Sorensen, 1987; Holten *et al.*, 2012), and (ii) the tension induced by ice is approx. 1.16 MPa·K⁻¹ (Hansen & Beck, 1988). Ice nucleation in the basal part of the branch thus can pull liquid sap from the distal end, which redistributes water toward nucleation sites (Ball *et al.*, 2006; Charra-Vaskou *et al.*, 2016), explaining the gradient in water potential and higher embolism observed under similar conditions (Mayr & Charra-Vaskou, 2007), and the higher number of UEs in branches than in any other part of the trunk. Nevertheless, during daytime, distal ends most likely thawed first and, hydraulically disconnected from the rest of the plant, would subsequently experience higher levels of drought stress and embolism compared to basal parts (Lemoine *et al.*, 1999). Such a spatial distribution of embolism within the plant may prevent embolism from spreading towards the base when the soil is frozen.

The present study reveals an enormous complexity in freezing dynamic and ice distribution within trees (Fig. 8). We showed that ice fronts may diverge into different directions within a stem or collide in other stem sections, which may cause separation of the hydraulic continuum
366 unless ice blockages dissolve during thawing. The combined use of several sensing
367 technologies enabled us to demonstrate not only contrasting ice propagation patterns, but also
368 the link between ice formation, diameter changes due to water shifts and ultrasonic activity
369 due to cavitation. These processes are highly relevant for plant hydraulics and freezing stress,
370 and their understanding is a prerequisite for studies on winter stress not only in timberline
371 species but also for temperate trees in general.

372

373 **Material and methods**

374 **Study site and tree description**

375 Study trees were naturally growing in Praxmar (Tyrol, Austria; 47°09′N; 11°07′E; 1680 m
376 a.s.l.) close to the timberline. Two Norway Spruce trees (Picea abies L. Karst), located on the
377 edge of the closed forest, were monitored during late winter. One tree was completely isolated
378 from surrounding trees (thereafter called tree 1), whereas the other one was located closer to
379 surrounding trees (thereafter called tree 2). Tree 1 was monitored repeatedly during four
380 successive years from 2012 until 2015, whereas tree 2 was only monitored during late winter
381 2015. Both trees were similar in dimension although growing during the four monitored
382 years: height from approx. 2.5 to 4 m, diameter at breast height from 5 to 10 cm. The
383 monitoring period ranged from early March until late April, at a period when frequent diurnal
384 freeze-thaw cycles are known to occur. At that time, a snow cover was still present and the
385 soil frozen. Freezing events were monitored with different sensors (thermocouples, UE
386 sensors, and dendrometers) at six positions along the main axis, from trunk base to top, and on
387 a side branch (Fig. 1). Depending on the availability of equipment, installations were made in
388 different combinations.

389 **Temperature measurements**
390 Xylem and air temperature were monitored using copper-constantan thermocouples connected
to a datalogger (1000 Series Squirrel Meter/Logger, Eltek, Cambridge, UK) at 30-60 s
391 interval. Thermocouples were installed in the xylem at 0.5-1 cm depth at 6 positions, along
392 the main trunk (apex: T₁ to base: T₅) and on a side branch (B), and attached using Terostat
393 putty (Henkel, Düsseldorf, Germany).
394

395 **Infrared imaging**

396 Surface temperature was recorded using a thermal infrared camera (Jenoptik VarioCam® HR,
397 InfraTec, Jena, Germany) at approx. 7 m distance from the trees. This camera is sensitive to
398 radiation at 7.5–14 µm wavelength of the electromagnetic spectrum with a temperature
399 resolution < 0.08 K. Thermal images (thermograms) were recorded at 0.025 m focal length,
400 resulting in 384 x 288 pixel images at approx. 0.01 m².pixel⁻¹ spatial resolution. Thermograms
401 were recorded every 5 seconds, and processed using IRBIS® 3 plus software (InfraTec, Jena,
402 Germany).
403

403 **Ultrasonic emission analysis**

404 UEs were monitored on Tree 1 with a 4-channel USB based system (1283 USB AEnode, 18-
405 bit A/D, 20 MHz) and 150 kHz 26 dB pre-amplified resonance sensors (PK15I; all
406 components: Physical Acoustics, Wolfegg, Germany) set to 20 dB threshold. On Tree 2, an
407 autonomous AE system device was installed, also equipped with four 150 kHz 26 dB pre-
408 amplified resonance sensors (PK15I; all components: Mistras, Sucy-en Brie, France).
409 Registration and analysis of ultrasonic events were performed with AEwin software vE.4.40
410 (Mistras Holdings Corp., Princeton, USA). Acoustic sensors were attached using spring-
411 loaded clamps at three positions along the trunk (T₂, T₃, T₅) and on a side branch (B; Fig. 1)
412 after removing approx. 1 cm² of bark and covering the exposed xylem with silicone grease to
413 ensure the acoustic coupling and prevent dehydration. Acoustic coupling was tested with lead
breaks (Hsu Nielsen method; Kalyanasundaram et al., 2007; Charrier et al., 2014b) at a
distance of 1 cm from each sensor, and sensors were re-installed when the signal amplitude
was below 90 dB.

417 Micro-dendrometers

418 Diurnal and longer-term variation in stem diameter was monitored using dendrometers on
419 both trees during late winter 2015. A PépiPIAF system (Forest Future, Nancy, France) was
420 installed in the upper third of the main axis (between T2 and T3, Fig. 1) of each tree. Each unit
421 contains a linear variable differential transformer (LVDT) sensor (sensitivity ±1 µm)
422 connected to a data logger recording at 5 min interval.

423 Supplemental material

Figure S1. Freezing patterns monitored via infrared imaging (A, B) and thermocouples (C, D).

425 Supplementary video S1 shows the temporal progression of differential surface temperatures
426 of a *Picea abies* tree and the surrounding snow cover, recorded by infrared imaging on March
427 14, 2015.

428 Supplementary video S2 shows an example of poikilothermic vs homeothermic regulation in
429 alpine winter conditions, recorded on the evening of March 3, 2011.

431 **Figure captions**

Figure 1. Measurement setup and sensor positions along a *Picea abies* tree.

Figure 2. Freezing patterns as observed via freezing exotherms along a *Picea abies* tree growing at the alpine timberline after sunset using thermocouples inserted into the xylem.

Left panels (A, C, E) show absolute temperatures during one evening. Right panels (B, D, F)
present differential thermal analysis (xylem - air temperature) during the same freezing
events. Arrows indicate the onset of exotherms at different sensor positions (B, T₁-T₅)
according to Fig. 1.

**Figure 3.** Spatial temperature patterns in a *Picea abies* tree growing at the alpine timberline at the time of freezing (after sunset; onset at approx. 17:55) monitored via infrared imaging. Letters (B, T₁-T₄) indicate sensor positions (see Fig. 1), arrows indicate additional positions with very pronounced changes. Note different time scales in the upper vs. lower row. Upper grey panels (A) show temperature changes over two hours within the crown. Lower panels (B) illustrate the period when xylem exotherms were detected (17:55 to 18:05). This timeframe thus corresponds to the onset of the freezing, and areas with pronounced events were highlighted in the thermograms.

**Figure 4.** Sequence of freezing exotherms observed using thermocouples (differential thermal analysis, xylem - air temperature; A) and cumulated ultrasonic emissions (UEs; B) in a *Picea abies* tree during a freezing event. Arrows indicate the onset of exotherms at different sensor positions (B, T₂, T₃, T₅).

**Figure 5.** Ultrasonic activity (A) and stem diameter changes (B) in a *Picea abies* tree during a freezing event. Temperatures are given as absolute (T₃, Air; A) and differential (xylem - air temperature; B) values. Stem diameter is given as differential with respect to initial diameter (i.e. Day 1, 12:00) to highlight dynamic changes. Vertical dashed lines indicate the onset of the exotherms as observed using thermocouple data.

**Figure 6.** Diurnal sequences of environmental conditions (air and xylem temperature, vapor pressure deficit; A), stem diameter changes, and ultrasonic activity (B) in a *Picea abies* tree during six days with five freeze-thaw cycles. Stem diameter is given as differential with
respect to initial diameter (i.e. Day 1, 12:00) to highlight dynamic changes. Vertical dashed lines indicate the onset of the freezing exotherms as observed by thermocouples.

**Figure 7.** Average ultrasonic activity (filled dots) and diameter shrinkage (white dots) observed per night (net radiation < 10 W.m⁻²) depending on average air temperature (A), or per day depending on average soil temperature (B). Average ultrasonic activities depending on diameter shrinkage (C) are presented with no significant difference between day (white diamonds) and night (filled diamonds). Note that y-scales are logarithmic; R² values apply to exponential (UEs activities), or linear fits (diameter shrinkage).

**Figure 8.** Generalized scheme of observed patterns and underlying water fluxes in a *Picea abies* tree during a freeze-thaw cycle in late winter. Initial equilibrated state (A), ice nucleation and propagation with associated water fluxes, stem shrinkage and ultrasonic emissions (B), ultrasonic activity after exotherm dissipation (C), stem diameter increase and developing water potential gradient during thawing (D), and stem shrinkage and high ultrasonic activity during daytime transpiration with frozen soil (E).

473

474

475

476

**Figure S1.** Freezing patterns monitored via infrared imaging (A, B) and thermocouples (C, D).

**Supplementary video 2.** Dynamic change of surface temperature in *Picea abies* and *Lepus europaeus* recorded by infrared image analysis at 1 s interval on March 3, 2011.
**Supplementary video 1.** Dynamic change of surface temperature in two *Picea abies* trees growing at the timberline, recorded by infrared image analysis at 5 s interval on March 14, 2015.

**Figure S1.** Freezing patterns monitored via infrared imaging (A, B) and thermocouples (C, D). Left panels (A, C) show absolute temperatures, right panels (B, D) show differential thermal analysis (xylem - air temperature) during the same freezing event. Arrows indicate the onset of exotherms at different sensor positions (B, T₁, T₃) according to Fig. 1.

**Supplementary video 1.** Dynamic change of surface temperature in two *Picea abies* trees growing at the timberline, recorded by infrared image analysis at 5 s interval on March 14, 2015. The video shows differential temperatures between 17:45 and 18:30, based on a reference frame recorded at 17:45. Warmer areas appear brighter, and areas where temperature was more than 1K above the reference temperature appear in red. Markers indicate the positions of the thermocouple sensors (T₁, T₂, T₃, T₄, B) and ultrasonic sensors (T₂, T₃, B).

**Supplementary video 2.** Dynamic change of surface temperature in *Picea abies* and *Lepus europaeus* recorded by infrared image analysis at 1 s interval on March 3, 2011. at approx. 19:15. This is a typical example of poikilothermic (*P. abies*) vs. homeothermic (*L. europaeus*) regulation in alpine winter conditions.

499
500 Acknowledgements

This work was supported by the Agence Nationale de la Recherche and FWF Fonds zur Förderung der Wissenschaftlichen Forschung (I826-B25 "Acoufreeze", TA, GC, KCV, SM).

MN is a recipient of a DOC-fellowship of the Austrian Academy of Sciences. We thank InfraTec for support and providing an IRBIS® plus software license to analyze and visualize infrared imaging data.

506

Table I. Spearman's correlation coefficient ρ between stem diameter variation, cumulated ultrasonic emissions observed during day and night periods and environmental variables i.e. minimum air temperature, minimum soil temperature and maximum vapor pressure deficit. Because of the particular local conditions, night-day shift was selected based on a threshold in net incoming radiation equal to 10 W·m⁻². Different symbols refer to P-value: *** P<0.001; ** P<0.01; * P<0.05; ns not significant.

513

<table><thead><tr><th></th><th></th><th>df</th><th>Minimum air temperature</th><th>Minimum soil temperature</th><th>Maximum vapor pressure deficit</th></tr></thead><tbody><tr><td rowspan="2">Night</td><td>Diameter change</td><td>33</td><td>-0.351*</td><td>0.325<sup>ns</sup></td><td>-0.877***</td></tr><tr><td>Ultrasonic emission</td><td>38</td><td>-0.753***</td><td>-0.168<sup>ns</sup></td><td>-0.387*</td></tr><tr><td rowspan="2">Day</td><td>Diameter change</td><td>33</td><td>-0.511**</td><td>-0.451**</td><td>0.273<sup>ns</sup></td></tr><tr><td>Ultrasonic emission</td><td>37</td><td>-0.219<sup>ns</sup></td><td>-0.575***</td><td>0.322*</td></tr></tbody></table>

514

515

516

517

518

519

520
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<figure><img src="image_3.png" /><figcaption>**Figure 1.** Measurement setup and sensor positions along a *Picea abies* tree.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Freezing patterns as observed via freezing exotherms along a *Picea abies* tree growing at the alpine timberline after sunset using thermocouples inserted into the xylem. Left panels (A, C, E) show absolute temperatures during one evening. Right panels (B, D, F) present differential thermal analysis (xylem - air temperature) during the same freezing events. Arrows indicate the onset of exotherms at different sensor positions (B, T₁ - T₅) according to Fig. 1.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Spatial temperature patterns in a *Picea abies* tree growing at the alpine timberline at the time of freezing (after sunset; approx. 17:55) monitored via infrared imaging. Letters (B, T₁-T₄) indicate sensor positions (see Fig. 1), arrows indicate additional positions with very pronounced changes. Note different time scales in the upper vs. lower row. Upper grey panels (A) show temperature changes over two hours within the crown. Lower panels (B) illustrate the period when xylem exotherms were detected (17:55, 17:58, 17:59, 18:05). This corresponds to the onset of the freezing, and areas with pronounced events where highlighted in the thermograms.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 4.** Sequence of freezing exotherms observed using thermocouples (differential thermal analysis, xylem - air temperature; A) and cumulated ultrasonic acoustic emissions (UEs; B) in a *Picea abies* tree during a freezing event. Arrows indicate the onset of exotherms at different sensor positions (B, T₂, T₃, T₅).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Ultrasonic activity (A) and stem diameter changes (B) in a *Picea abies* tree during a freezing event. Temperatures are given as absolute (T₃, Air; A) and differential (xylem - air temperature; B) values. Stem diameter is given as differential with respect to initial diameter (i.e. Day 1, 12:00) to highlight dynamic changes. Vertical dashed lines indicate the onset of exotherms as observed using thermocouple data.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 6.** Diurnal sequences of environmental conditions (air and xylem temperature, vapor pressure deficit; A), stem diameter changes, and ultrasonic activity (B) in a *Picea abies* tree during six days with five freeze-thaw cycles. Stem diameter is given as differential with respect to initial diameter (i.e. Day 1, 12:00) to highlight dynamic changes. Vertical dashed lines indicate the onset of the freezing exotherms as observed by thermocouples.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Figure 7.** Average ultrasonic activity (filled dots) and diameter shrinkage (white dots) observed per night (net radiation < 10 W·M⁻²) depending on average air temperature (A), or per day depending on average soil temperature (B). Average ultrasonic activities depending on diameter shrinkage (C) are presented with no significant difference between day (white diamonds) and night (filled diamonds). Note that 30-fold increase in main R² values apply to exponential (UE activities) or linear fits (diameter shrinkage).</figcaption></figure>

Copyright © 2017 American Society of Plant Biologists. All rights reserved.
<figure><img src="image_10.png" /><figcaption>**Figure 8.** Generalized scheme of observed patterns and underlying water fluxes in a *Picea abies* tree during a freeze-thaw cycle in late winter. Initial equilibrated state (A), ice nucleation and propagation, with associated water fluxes, stem shrinkage and ultrasonic emissions (B), ultrasonic activity after exotherm dissipation (C), stem diameter increase and developing water potential gradient during thawing (D), and stem shrinkage and high ultrasonic activity during daytime transpiration with frozen soil (E).</figcaption></figure>
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<figure><img src="image_11.png" /></figure>

**Figure S1.** Freezing patterns monitored via infrared imaging (A, B) and thermocouples (C, D). Left panels (A, C) show absolute temperatures, right panels (B, D) show differential thermal analysis (xylem - air temperature) during the same freezing event. Arrows indicate the onset of exotherms at different sensor positions (B, T₁, T₃), according to Fig. 1.