Winter Embolism and Recovery in the Conifer Shrub Pinus mugo L.

: Research Highlights: Pronounced winter embolism and recovery were observed in the Alpine conifer shrub Pinus mugo L. Data indicated that the hydraulic courses and underlying mechanism were similar to timberline trees. Background and Objectives: At high elevation, plants above the snow cover are exposed to frost drought and temperature stress during winter. Previous studies demonstrated winter stress to induce low water potentials ( Ψ ) and signiﬁcant xylem embolism (loss of conductivity, or LC) in evergreen conifer trees, and recovery from embolism in late winter. Here, we analyzed xylem hydraulics and related structural and cellular changes in a conifer shrub species. Materials and Methods: The uppermost branches of Pinus mugo shrubs growing at the Alpine timberline were harvested over one year, and the Ψ , water content, LC, proportion of aspirated pits, and carbohydrate contents were analyzed. Results: Minimum Ψ ( − 1.82 ± 0.04 MPa) and maximum LC (39.9% ± 14.5%) values were observed in mid and late winter, followed by a recovery phase. The proportion of aspirated pits was also highest in winter (64.7% ± 6.9% in earlywood, 27.0% ± 1.4% in latewood), and decreased in parallel with hydraulic recovery in late winter and spring. Glucose and fructose contents gradually decreased over the year, while starch contents (also microscopically visible as starch grains in needle and stem tissues) increased from May to July. Conclusions: The formation and recovery of embolism in Pinus mugo were similar to those of timberline trees, as were the underlying mechanisms, with pit aspiration enabling the isolation of embolized tracheids, and changes in carbohydrate contents indicating adjustments of osmotic driving forces for water re-distribution. The e ﬀ ects of future changes in snow cover regimes may have pronounced and complex e ﬀ ects on shrub-like growth forms, because a reduced snow cover may shorten the duration of frost drought, but expose the plants to increased temperature stress and impair recovery processes.


Introduction
At high elevation, plants, unless protected by the snow cover, are exposed to high stress intensities during winter. They have to cope with temperature stress (i.e., low minimum temperatures and high temperature fluctuations) as well as winter drought. Especially evergreen trees were demonstrated to experience frost drought, because water is lost via (cuticular) transpiration over the crown, while the frozen soil blocks water uptake for months [1]. Consequently, low water potentials (Ψ) develop, which, in combination with frequent freeze-thaw events, can lead to the formation of xylem embolism.
Embolism blocks the water transport and thus the water supply of distal tissues. On drought, low Ψ causes air entry into xylem conduits via pits ("air seeding"; [2]), and disruption of the metastable water

Plant Material
This study was performed on knee pine (Pinus mugo Turra) growing at Birgitz Köpfl, Tyrol, Austria (2035 m, 47°11'N/11°19'E). For measurements, the uppermost, sun-exposed branches of shrubs (height up to 2.5 m) growing in a northwest exposed slope were used. For logistic reasons, samples harvested at the timberline were sometimes stored at −20 °C until taking measurements of sugar/starch contents and anatomy.
Measurements during winter and spring were performed in parallel with a study on Picea abies [1]. Climate data (daily minimum, mean and maximum air temperatures, daily sum of precipitation) of a nearby weather station (Mt. Patscherkofel, 2.252 m) were provided by ZAMG Zentralanstalt für Meteorologie und Geodynamik, Austria.

Seasonal Course
Samples were harvested from November 2010 to November 2011 in ca. monthly intervals between 11:00 and 14:00, respectively. Three shrubs were randomly selected and one branch per shrub was harvested, whereby in winter, branches above the snow cover were used. First, end twigs (ca. 10 cm) of branches were sampled and enclosed in plastic bags for transport to the laboratory and analysis of water potential (Ψ; see Section 2.3) and needle sugar/starch content. Second, branches (up to 1 m in length, basal diameter up to 1.5 cm, age up to 10 years) were cut and enclosed in plastic bags. Main stems of these branches were used for analysis of the loss of conductivity (LC), proportion of aspirated pits, and xylem and phloem starch content. In addition, needles were sampled for microscopic analyses (sugar/starch, proportion of aspirated pits).

Water Potential (Ψ)
Ψ measurements were made directly after field trips. For the analysis of Ψ, terminal segments of three side twigs (length up to 10 cm) per selected branch were cut, tightly wrapped in plastic bags,

Plant Material
This study was performed on knee pine (Pinus mugo Turra) growing at Birgitz Köpfl, Tyrol, Austria (2035 m, 47 • 11'N/11 • 19'E). For measurements, the uppermost, sun-exposed branches of shrubs (height up to 2.5 m) growing in a northwest exposed slope were used. For logistic reasons, samples harvested at the timberline were sometimes stored at −20 • C until taking measurements of sugar/starch contents and anatomy.
Measurements during winter and spring were performed in parallel with a study on Picea abies [1]. Climate data (daily minimum, mean and maximum air temperatures, daily sum of precipitation) of a nearby weather station (Mt. Patscherkofel, 2.252 m) were provided by ZAMG Zentralanstalt für Meteorologie und Geodynamik, Austria.

Seasonal Course
Samples were harvested from November 2010 to November 2011 in ca. monthly intervals between 11:00 and 14:00, respectively. Three shrubs were randomly selected and one branch per shrub was harvested, whereby in winter, branches above the snow cover were used. First, end twigs (ca. 10 cm) of branches were sampled and enclosed in plastic bags for transport to the laboratory and analysis of water potential (Ψ; see Section 2.3) and needle sugar/starch content. Second, branches (up to 1 m in length, basal diameter up to 1.5 cm, age up to 10 years) were cut and enclosed in plastic bags. Main stems of these branches were used for analysis of the loss of conductivity (LC), proportion of aspirated pits, and xylem and phloem starch content. In addition, needles were sampled for microscopic analyses (sugar/starch, proportion of aspirated pits).

Water Potential (Ψ)
Ψ measurements were made directly after field trips. For the analysis of Ψ, terminal segments of three side twigs (length up to 10 cm) per selected branch were cut, tightly wrapped in plastic bags, and transported to the laboratory within 1-2 h. Then, the Ψ of samples was measured with a Scholander apparatus (Model 1000, PMS, USA), whereby the entire end twig was sealed into the pressure chamber before the pressure was slowly increased until the water front was visible at the cut stem surface. The discrimination of water and resin was possible due to different viscosities and bubble sizes. Ψ values were averaged per branch.

Loss of Conductivity (LC) and Water Content (WC)
Samples for LC measurements were stored and wrapped in plastic bags in a fridge at ca. 10 • C until the following day at a maximum. LC was measured with a Xyl'em system (Bronkhorst, France) on three subsamples and averaged per branch. Segments (length~4 cm, xylem diameter~0.8 cm) of the main stem were cut under water, and LC was determined by measuring the increase in hydraulic conductivity after the removal of xylem embolism by repeated high-pressure flushes [34]. The sample preparation and measurement procedure followed a previously described protocol [11]: Flushing (at 80 kPa) and conductivity measurements (at 4 kPa) were done with distilled, filtered (0.22 µm), and degassed water containing 0.005% (v/v) "Micropur" (Katadyn Products Inc., Wallisellen, Switzerland) to prevent microbial growth. Flushing was repeated until measurements showed no further increase in conductivity, and the loss of conductivity was calculated as LC = (1 − K min /K max ) × 100, where K min is the initial conductivity, and K max is the maximal conductivity.
WC was measured gravimetrically on additional segments (length ca. 2 cm) cut from the main stem of samples (ME2355, Sartorius, Götingen, Germany). Bark and wood were separated, and the fresh weight was determined before samples were dried at 80 • C in and oven (48 hours) and re-weighted.

Anatomical Analysis
Fresh branch segments, and needles were placed directly into 100% ethanol in order to avoid pit aspiration [35,36]. Samples remained in 100% ethanol for 48 h, and were thereafter embedded in Technovit 7100 (Heraeus Kulzer, Germany). Semi-thin transverse sections (1-2 µm) were made on a rotary microtome (RM2235 Leica, Germany). Sections were thereafter stained for 3 min with Toluidine blue (Merck, Germany) solution (0.03 g/100 mL tap water (pH 7)), washed in distilled water, dehydrated in 99% alcohol, and mounted in Euparal (Carl Roth, Germany). The staining of starch grains was performed with Lugol´s solution (Fluka, Sigma-Aldrich, USA). Lugol´s solution is a solution of 2 g of potassium iodide with 1 g of iodine in distilled water. Samples were stained for 2min, washed with distilled water, and embedded in glycerol.
Starch grains and bordered pits were observed with a Leica CTR6000 microscope equipped with a digital camera (DMC2900 Leica, Germany). Pits in the xylem of needles and in the sapwood were classified as "closed" if the pit membrane was aspirated (Figure 2a,b) or as "open" if the pit membrane was positioned in the middle of the pit chamber (Figure 2c,d). Toluidine blue stains the torus of the pit in dark purple-dark blue, which gives a strong contrast to the turquoise-stained lignified pit chamber walls and secondary cell walls of the tracheids [1]. The classification of pit aspiration in order to assess the proportion of aspirated pits in sapwood was done for the earlywood and latewood of the latest three annual rings within a tangential window of 2-3 mm on 2-3 specimens from each tree. Regarding latewood, only pits in the last formed five cell rows close to the ring border were analyzed ( Figure 2a). The screening of pit aspiration in earlywood was done mainly in the first formed 10 cell rows. In total, 7163 bordered pits were analyzed: on average, 512 pits per tree.
showed no further increase in conductivity, and the loss of conductivity was calculated as LC = (1−Kmin/Kmax) × 100, where Kmin is the initial conductivity, and Kmax is the maximal conductivity.
WC was measured gravimetrically on additional segments (length ca. 2 cm) cut from the main stem of samples (ME2355, Sartorius, Götingen, Germany). Bark and wood were separated, and the fresh weight was determined before samples were dried at 80 °C in and oven (48 hours) and re-weighted.

Glucose, Fructose, and Starch Content
Analyses were performed for all samples harvested between November 2010 and August 2011. The needles were cut from branches, and from the main stem, and xylem and phloem samples were separated. Dried and finely ground plant material was incubated two times with 80% ethanol (v/v) at 75 • C with polyvinylpyrrolidon (PVP 40, Sigma Chemicals, USA) added. The supernatants containing the low molecular weight sugars were collected, the solvent was evaporated, and the residue was dissolved in citrate buffer pH 4.0. After dissociating the sucrose with invertase (β-fructosidase), the content of glucose was determined photometrically [37]. Fructose was converted to glucose with isomerase (phosphoglucose isomerase) and quantified using a standard test (Enzytec E1247, r-biopharm, Germany) measuring the absorption of NADPH [38] at 340 nm. The precipitate containing the starch was incubated with sodium hydroxide (0.5 M, 1 h at 60 • C), neutralized with hydrochloric acid, treated with amylase (amyloglucosidase) to break down the starch to glucose, and measured as described (Enzytec E1210, r-biopharm, Germany).

Statistics
Values were averaged per branch, which is the statistical unit throughout the study, and are given as the mean ± SE. Differences were tested with Student's t test and correlations with Pearson's coefficient were determined at p = 0.05.

Water Potential and Embolism
During summer months (May-September; Figure 3b), Ψ varied between −1 and −1.5 MPa. Note that these measurements were taken around midday on sunny days and thus reflect values during transpiration. In winter, a decrease to −1.82 ± 0.04 MPa (22 February), followed by a steep increase to −0.34 ± 0.04 MPa (4 April), was observed. Values before (22 February) and after (19 May) this phase of intense winter drought significantly differed from peak Ψ in midwinter (22 March). The course of Ψ followed the course of the water content of the bark and (less) to that of the xylem, although no significant correlation was observed. LC was 15% at a maximum from November 2010 to the end of 2010 and from April 2011 to November 2011 (Figure 3c). In winter months, a maximum of 39.9% ± 14.5% was reached at 22 March, and significant differences between midwinter (22 February) and late winter values (19 May) were found.

Proportion of Aspirated Pits
The proportion of aspirated pits was overall higher in earlywood than in latewood (Figure 3d). The highest proportion of aspirated pits was observed on 22 February (64.7% ± 6.9% in earlywood (Figure 2b), 27.0% ± 1.4% in latewood), after which pit aspiration gradually decreased (17.5% ± 4.1% in earlywood, 3.6% ± 3.2% in latewood on 19 May). The strongest decrease was observed between 4 April and 19 May (significant differences in earlywood). A high proportion of latewood pits did not aspirate, even during winter time (Figure 2a). The lowest proportion of aspirated pits could be found from May to July (Figure 2c,d, Figure 3d). Although Figure 3 indicates a parallel decrease in LC and proportion of aspirated pits, due to the limited number of data points, no significant correlation was observed.
In needles, no screening for the proportion of aspirated pits could be made, because in each transverse section of the vascular bundles, only a few number of bordered pits could be found. However, we have hints that the bordered pits of the needle xylem are as well closed in winter (Figure 4a,b) and open in summer (Figure 4c,d) until-at least-early September (Figure 4e).

Glucose, Fructose and Starch Content
Most pronounced courses in carbohydrate contents were found in needles (Figure 5a): While glucose and fructose concentrations gradually decreased from November 2010 (3.3% ± 0.5% DW glucose, 2.9% ± 0.4% DW fructose) to August 2011 (1.5% ± 0.3% DW glucose, 1.1% ± 0.2% DW fructose), starch contents were low until May 2010, peaked in July 2011 (9.3% ± 1.1% DW), and then decreased to 1.4% ± 0.8% DW (August 2011). July values were significantly higher than those in March and August. Annual course patterns were similar but less clear in stem sapwood and secondary phloem, with the highest starch contents reached in July (Figure 5b,c). Histochemical observation of the starch grains ( Figure 6) in needles and secondary phloem confirmed the results of the chemical analyses. Firstly, a lower number of starch grains were found in the living cells of the secondary phloem, i.e., parenchyma cells of the rays and of the tangential parenchyma bands between the sieve cells (Figure 6a), compared to the needles (Figure 6b,c) and secondly, the maximum amount of starch was present in July 2011 in both organs. Starch grains were detectable until November 2011, but not in the period from 21 December 2010 until 22 March 2011. In midwinter and early spring, when starch was absent in the needles (Figure 7a), several white-colored vesicles were found in the mesophyll cells, and the cells of the endodermis contained small translucent (hydrophobic) droplets. During late spring and summer, no such droplets were found in the endodermis (Figure 7b); instead, the cells were filled with quite big starch grains. Starch  Histochemical observation of the starch grains ( Figure 6) in needles and secondary phloem confirmed the results of the chemical analyses. Firstly, a lower number of starch grains were found in the living cells of the secondary phloem, i.e., parenchyma cells of the rays and of the tangential parenchyma bands between the sieve cells (Figure 6a), compared to the needles (Figure 6b,c) and secondly, the maximum amount of starch was present in July 2011 in both organs. Starch grains were detectable until November 2011, but not in the period from 21 December 2010 until 22 March 2011. In midwinter and early spring, when starch was absent in the needles (Figure 7a), several white-colored vesicles were found in the mesophyll cells, and the cells of the endodermis contained small translucent (hydrophobic) droplets. During late spring and summer, no such droplets were found in the endodermis ( Figure 7b); instead, the cells were filled with quite big starch grains. Starch grains in the endodermis were much bigger than those in the parenchyma cells of the mesophyll or near the vascular bundles (Figure 6b). The Casparian strip of the endodermis is impregnated with lignin, as indicated by its turquoise-greenish color when stained with toluidine blue.

Discussion
Winter stress induced embolism in Pinus mugo, as observed in other conifers growing at high elevation. Pinus mugo also showed recovery during late winter and spring, and findings indicate that underlying mechanisms were similar to those found in timberline trees: pit aspiration probably enabled the isolation of embolized tracheids until their functionality was re-established (Figure 3d), and changes in carbohydrate contents demonstrate cellular activity during late winter, which might enable osmotic adjustment to create driving forces for water re-distribution. However, in contrast to our hypothesis, the annual course in water potential (Ψ), loss of conductivity (LC), and related structural parameters were not more complex than that in trees, despite the shrub habit of Pinus mugo and respective more snow coverage.
The maximum LC in Pinus mugo reached during the study period was about 40% (Figure 3c), which is remarkably similar to the LC in Picea abies (43.4% ± 1.6%; [1]) observed in the same winter season and at the same study site. Accordingly, the minimum Ψ was about −1.8 MPa in Pinus mugo ( Figure 3b) and −1.69 ± 0.11 MPa in Picea abies. This indicates that conifer shrubs and trees were exposed to identical winter drought stress, and that the more frequent cover by snow did not protect shrubs from dehydration during winter months. This may be due to the following two explanations: First, note that uppermost branches were harvested during winter. These were probably the branches experiencing the most stress and being protected by snow for relatively short periods only. [33] already demonstrated that branches permanently covered by snow develop only moderate Ψ

Discussion
Winter stress induced embolism in Pinus mugo, as observed in other conifers growing at high elevation. Pinus mugo also showed recovery during late winter and spring, and findings indicate that underlying mechanisms were similar to those found in timberline trees: pit aspiration probably enabled the isolation of embolized tracheids until their functionality was re-established (Figure 3d), and changes in carbohydrate contents demonstrate cellular activity during late winter, which might enable osmotic adjustment to create driving forces for water re-distribution. However, in contrast to our hypothesis, the annual course in water potential (Ψ), loss of conductivity (LC), and related structural parameters were not more complex than that in trees, despite the shrub habit of Pinus mugo and respective more snow coverage.
The maximum LC in Pinus mugo reached during the study period was about 40% (Figure 3c), which is remarkably similar to the LC in Picea abies (43.4% ± 1.6%; [1]) observed in the same winter season and at the same study site. Accordingly, the minimum Ψ was about −1.8 MPa in Pinus mugo ( Figure 3b) and −1.69 ± 0.11 MPa in Picea abies. This indicates that conifer shrubs and trees were exposed to identical winter drought stress, and that the more frequent cover by snow did not protect shrubs from dehydration during winter months. This may be due to the following two explanations: First, note that uppermost branches were harvested during winter. These were probably the branches experiencing the most stress and being protected by snow for relatively short periods only. [33] already demonstrated that branches permanently covered by snow develop only moderate Ψ and LC. Second, trees can store a lot of water in the trunk, which may buffer water losses induced by frost drought. [31] found water storage to increase and drought effects to decrease with tree size. The small branches of Pinus mugo cannot store larger amounts of water and thus Ψ decreased, even when drought stress was lower than in neighboring trees. In the branches harvested for this study, we could also show that even the bordered pits of the xylem from the needle vascular bundles can be aspirated during winter time (Figure 4a,b) in order to prevent the further embolism of adjacent tracheids. Relatively short-term snow covers on upper branches may also explain why we did not observe complex sequences of embolism formation and recovery but, as in trees [7,[9][10][11]14,15], a period of embolism formation during midwinter and of recovery during late winter and spring. Similar to findings in Picea abies [1], embolism formation and repair thereby corresponded to changes in pit aspiration and carbohydrate contents.
The course of the proportion of aspirated pits followed the course of LC during the recovery period with decreasing LC and the opening of pits from late winter onwards (Figure 3c,d). In contrast, the proportion of aspirated pits was already high from the beginning of winter, while LC progressively increased. This indicates that some pits closed independently of embolism formation, maybe due to early frost events. Accordingly, the correlation (earlywood pits r 2 = 0.25) of the proportion of aspirated pits versus LC was weaker than that observed in Picea abies [1]. Overall, earlywood tracheids showed a higher proportion of aspirated pits, while latewood pits remained open more frequently. It remains to be studied whether this is due to lower embolism or less efficient pit aspiration in latewood [35]. As earlywood tracheids and their pits are hydraulically much more relevant than latewood tracheids, the findings again underline that the special conifer pit architecture [28,39] plays a central role for conifer hydraulic safety and pit aspiration, as well as probably for embolism recovery in secondary xylem (Figure 2), and even in the xylem of the vascular bundles of the needles (Figure 4).
Observed recovery also corresponded to changes in carbohydrate contents ( Figure 5). Similar to Picea abies [1], a first peak in starch concentrations was observed in late winter (4 April), indicating relevant redistributions of carbohydrates within the plants. However, starch was not present year-round in Pinus mugo secondary phloem and needles. In the period from December until March, lipid droplets were found, especially in the endodermis cells (Figure 7a). According to [40], fats or lipid droplets are present the whole year; in some species, they are the only visible winter food reserve. Such species, including mainly diffuse porous angiosperm, but also the genus Pinus, are termed as "fat trees" [41,42]. In our study, starch grains were not present until the end of March ( Figure 6). It has to be considered that the first changes in starch contents were observed when the snow cover was still present and the plants did not have access to soil water. However, the link between changes in carbohydrate contents and xylem refilling has yet to be demonstrated. According to [22], it is likely that carbohydrates are required to create an osmotic force, which enables pulling water into water films on the cell walls of embolized conduits also; see [43][44][45]. However, we did not observe relevant changes in glucose or fructose during recovery, probably because the formation of osmotic active sugars occurs locally and in comparably low overall concentrations. Unfortunately, a separate analysis of tracheid contents, as in the much bigger Populus [44] vessels, is hardly possible in conifers. The remarkable drop in starch content in late summer (Figures 5 and 6) indicates the beginning of transition to lipid pools and the displacement of storage pools to stem and roots.
Overall, embolism and refilling in Pinus mugo were surprisingly similar to the seasonal patterns observed in other conifers. The relevance of snow cover protection for both embolism formation and repair complicates predictions of the winter stress situation under future climate: in the Alps, the temperature increase was above the global average during the past decades [46,47], so that reductions in snow cover heights and durations have to be expected in future. An estimation of future precipitation changes is difficult, as the mountain terrain is complex. However, some studies for instance indicate an increase of spring droughts [48][49][50]. A reduced snow cover may reduce the time of blocked access to soil water and thus reduce frost drought. At the same time, the lack of snow protection may expose the plants to increased temperature amplitudes and freeze-thaw cycles, which can also induce embolism (see introduction). Furthermore, a lack of snow cover in late winter may hinder refilling processes, when water cannot be taken up over the branch surface. Shrub-like growth forms, such as Pinus mugo, might be especially affected by changes in snow cover regimes, but more studies are required to unravel the significance and dynamics of processes during embolism formation and recovery.