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. 2022 Jan 31;189(1):204–214. doi: 10.1093/plphys/kiac034

Hydraulic vulnerability segmentation in compound-leaved trees: Evidence from an embolism visualization technique

Jia Song 1,2,3, Santiago Trueba 4, Xiao-Han Yin 5, Kun-Fang Cao 6, Timothy J Brodribb 7, Guang-You Hao 8,✉,†
PMCID: PMC9070814  PMID: 35099552

Abstract

The hydraulic vulnerability segmentation (HVS) hypothesis implies the existence of differences in embolism resistance between plant organs along the xylem pathway and has been suggested as an adaptation allowing the differential preservation of more resource-rich tissues during drought stress. Compound leaves in trees are considered a low-cost means of increasing leaf area and may thus be expected to show evidence of strong HVS, given the tendency of compound-leaved tree species to shed their leaf units during drought. However, the existence and role of HVS in compound-leaved tree species during drought remain uncertain. We used an optical visualization technique to estimate embolism occurrence in stems, petioles, and leaflets of shoots in two compound-leaved tree species, Manchurian ash (Fraxinus mandshurica) and Manchurian walnut (Juglans mandshurica). We found higher (less negative) water potentials corresponding to 50% loss of conductivity (P50) in leaflets and petioles than in stems in both species. Overall, we observed a consistent pattern of stem > petiole > leaflet in terms of xylem resistance to embolism and hydraulic safety margins (i.e. the difference between mid-day water potential and P50). The coordinated variation in embolism vulnerability between organs suggests that during drought conditions, trees benefit from early embolism and subsequent shedding of more expendable organs such as leaflets and petioles, as this provides a degree of protection to the integrity of the hydraulic system of the more carbon costly stems. Our results highlight the importance of HVS as an adaptive mechanism of compound-leaved trees to withstand drought stress.

The long-proposed hypothesis that more terminal organs have greater vulnerability to drought-induced embolism was tested and proved in shoots of compound-leaved trees using an optical technique.

Introduction

Water transport from roots to leaves within the soil–plant–atmosphere continuum occurs under negative pressure (Sperry et al., 2002), generating a water potential gradient throughout the plant (Dixon and Joly, 1895; Dixon, 1914). This water transport system is sensitive to drought, which can induce high xylem tensions provoking the formation of xylem embolisms, and ultimately leading to hydraulic failure (Tyree and Zimmermann, 2002). Plant organs vary greatly in water transport capacity and vulnerability to embolism during drought (Choat et al., 2005). The hydraulic segmentation hypothesis refers to the observation that proximal organs (i.e. stem) and distal organs (i.e. leaves) are separated by large hydraulic resistors, leading to a segmented water potential reduction along the plant hydraulic pathway (Zimmermann, 1978; Gates, 1980; Yazaki et al., 2010; Liu et al., 2015; Merine et al., 2015). The hydraulic segmentation hypothesis further developed into the hydraulic vulnerability segmentation (HVS) hypothesis, which suggests that distal organs are more vulnerable to drought-induced embolism than proximal stems (Choat et al., 2005; Hao et al., 2008; Zhang et al., 2009; Johnson et al., 2011; Scholz et al., 2014; Nolf et al., 2015; Zhu et al., 2016). In these regards, during seasonal and unpredictable drought stresses, some species might derive benefit from dropping their distal units (leaves), thereby reducing water loss and protecting the more carbon costly stems from catastrophic hydraulic failure and diebacks (Bucci et al., 2012; Pivovaroff et al., 2014). However, there is an obvious knowledge gap in our understanding of the variation in hydraulic vulnerability within the hydraulic system of individuals and species during drought, especially across different plant organs such as leaf laminas, petioles, and stems (Creek et al., 2018).

Vulnerability segmentation might be particularly important for trees living in habitats with marked seasonal droughts and seems to be particularly relevant in compound-leaved tree species (Gates, 1980; Tyree et al., 1993; Yazaki et al., 2010; Liu et al., 2015; Merine et al., 2015). Compound-leaved tree species are found to be more common in warmer and arid or semi-arid environments (Givnish, 1978; Stowe and Brown, 1981). Moreover, it has been suggested that compound-leaved tree species show rapid growth rates during favorable environmental conditions and often shed their leaves when facing unfavorable conditions (Gates, 1980; Niinemets, 1998). Indeed, an important adaptation of compound-leaved tree species is their habit of shedding entire leaves (consisting of leaflets and petioles) during drought, thereby protecting the more carbon costly stems (Givnish, 1978; Stowe and Brown, 1981; Yazaki et al., 2010; Merine et al., 2015). The petiole of compound-leaved tree species, particularly the ones with large leaves, have highly effective water transport rates permitting higher shoot hydraulic conductance in these species compared to sympatric tree species with simple leaves (Song et al., 2018; Yang et al., 2019), and allowing a high photosynthetic capacity when water is available (Rosati et al., 2006; Tulik et al., 2010). High hydraulic conductance and photosynthetic rates might allow compound-leaved tree species to have fast growth rates in sufficient water conditions (Malhado et al., 2010; Yang et al., 2019), but this may imply lower resistance to drought-induced hydraulic failure as a tradeoff of high hydraulic conductance. Thus, the vulnerability segmentation in the hydraulic system of shoots, which protects the main parts of the plant and minimizes the damage during water deficit, could be an effective strategy in compound-leaved tree species to cope with unpredictable sporadic drought stresses. Compound-leaved tree species therefore represent an interesting system for testing the vulnerability segmentation hypothesis.

A prerequisite for investigating HVS is to measure vulnerability curves accurately across the different organs of the plant hydraulic system in the same individual plants. Many efforts have been made in recent years to accurately quantify xylem embolism using reliable techniques to construct vulnerability curves; however, a detailed exploration of vulnerability segmentation across all the components of whole-shoots has been mainly prevented by major technical limitations (Cochard et al., 2013; Torres-Ruiz et al., 2017; Brodribb 2017). Vulnerability curves are mostly measured in stems, whereas data for other components of shoots such as leaflets and petioles are much less abundant, making it difficult to compare embolism vulnerability across different organs (Rodriguez-Dominguez et al., 2018; Wason et al., 2018). Even in studies with both stem and leaf vulnerability curves conducted, these vulnerability curves are often obtained using different techniques, compromising comparative accuracy (Hao et al., 2013a; Liu et al., 2015). Consequently, the degree of segmentation between all the components of a shoot, and how the vulnerability segmentation across organs is related to hydraulic adaptation and hydraulic safety remains unclear.

In compound-leaved tree species, vulnerability curves of different organs have been constructed using different techniques (Liu et al., 2015), leading to a potential lack of comparability due to the inconsistency of the employed methods. In addition, some methods for vulnerability curve construction might have limitations when applied under certain conditions, for instance, artifacts could result from the air-injection and centrifuge methods when applied to species bearing long vessels (Sperry et al. 2012; Wheeler et al., 2013; Rockwell et al., 2014; Pivovaroff et al. 2016). In a previous study, we found that compound-leaved tree species tend to have a ring-porous wood with long vessels as compared with their co-occurring single-leaved counterparts that dominantly have diffuse-porous wood (Yang et al., 2019). Therefore, an adapted method is needed to quantify xylem embolism and to construct vulnerability curves in the different organs of compound-leaved tree species with long vessels. In this context, a single nondestructive method that can be applied across all plant organs with high accuracy would improve our understanding of xylem vulnerability with respect to the segmentation hypotheses. The optical visualization technique is a recent, nondestructive technique that can be used to investigate embolism formation among distinct organs (Brodribb et al., 2016a, 2016b, 2017), offering an ideal technique to test the HVS hypothesis across different plant organs in compound-leaved tree species.

Here, we examine the variation in xylem vulnerability across organ types in whole-shoots of Manchurian ash (Fraxinus mandshurica) and Manchurian walnut (Juglans mandshurica), two compound-leaved tree species that are dominant in the temperate forest of Northeast (NE) China. According to a previous study, compound-leaved tree species, particularly the ones with large leaves such as F. mandshurica and J. mandshurica, show higher hydraulic conductance and increased efficiency of carbon assimilation than single-leaved tree species growing in a common garden, which were hypothesized as features leading to greater degrees of vulnerability segmentation (Song et al., 2018; Yang et al., 2019). Moreover, although both species occur in relatively humid environments, there is a possibility of experiencing severe droughts during the growing season (Fang et al., 2016), and HVS can therefore be an important protective mechanism to minimize risks during such unpredictable drought events. Given that both F. mandshurica and J. mandshurica have much wider and longer vessels than their single-leaved counterparts (Yang et al., 2019), it is particularly difficult to produce vulnerability curves accurately using traditional methods due to potential artifacts induced by the presence of open vessels. We therefore used the embolism visualization technique to estimate drought vulnerability across organs. We hypothesized that the proximal stems are more resistant to embolism than petioles and leaflets, and that stems might operate with a larger safety margin than leaflets and petioles to protect the more carbon costly organs.

Results

By applying the optical visualization technique across different parts of shoots (Figure 1), we found differences in the timing of embolism onset in leaf veins, petioles, and stems from the same individual, with embolism occurring earlier in leaflets and petioles than in stems (Supplemental Movies S1–S3). Significant differences in mean P50 (Supplemental Table S1) were recorded between leaflets, petioles and stems in both F. mandshurica and J. mandshurica (Figure 2), with stems (P50 = −3.10 ± 0.02 MPa in F. mandshurica; P50 = −3.65 ± 0.26 MPa in J. mandshurica) having a more negative P50 than petioles (P50 = −2.09 ± 0.02 MPa in F. mandshurica, P50 = −2.67 ± 0.03 MPa in J. mandshurica) and leaflets (P50 = −1.98 ± 0.08 MPa in F. mandshurica, P50 = −2.10 ± 0.09 MPa in J. mandshurica). Our data therefore show that leaflets and petioles are more prone than stems to develop xylem embolism under similar drought stress conditions.

Figure 1.

Figure 1

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The measurement setup for the embolism optical visualization technique and images showing the spatiotemporal progressive embolism formation in branch hydraulic systems of Juglans mandshurica. A, Diagram showing the positions of different measurements on an excised branch with compound leaves, (B) high-resolution images of leaflet, petiole, and stem xylem from the series of images taken during the dehydration process, (C) change of xylem water potential continuously monitored using a stem psychrometer, (D) spatiotemporal progressive embolism formation in the leaflet, petiole, and stem xylems. The color scale bar shows the labeling of xylem water potentials recorded at different cavitation events. Images for leaflets were taken using the transmission mode of the flatbed scanner while the reflective mode was used for scanning the xylem of petioles and stems.

Figure 2.

Figure 2

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Vulnerability curves constructed for leaflets, petioles, and stems using the optical visualization technique in Fraxinus mandshurica and Juglans mandshurica. Cumulative area (relative to the total) of cavitated vessels as a function of water potential in xylems of (A and B) leaflets, (C and D) petioles, and (E and F) stems for the two studied compound-leaved tree species. Solid red lines indicate the mean vulnerability curve fitted using a Sigmoid function and the 95% confidence intervals (n = 3). Light black lines show individual vulnerability curves. The dotted vertical lines show mean air-entry water potential (Pe), which is defined as the xylem water potential corresponding to 12% embolism in each part of shoot. Dashed vertical lines show mean xylem water potential responsible for 50% cumulative embolism (P50).

Significant differences in the mean water potential at the initiation of embolism formation (Pe) were observed when comparing leaflets, petioles, and stems (Supplemental Table S1). The water potential values of embolism initiation in stems were more negative (Pe = −1.42 ± 0.08 MPa in F. mandshurica, Pe = −1.39 ± 0.06 MPa in J. mandshurica), than in petioles (Pe = −0.62 ± 0.001 MPa in F. mandshurica, Pe = −0.88 ± 0.02 MPa in J. mandshurica) and leaflets (Pe = −0.84 ± 0.02 MPa in F. mandshurica, Pe = −0.75 ± 0.03 MPa in J. mandshurica) from the same individual (Figure 2). However, Pe did not significantly differ between petioles and leaflets in both species. We also observed variation in embolism propagation within the leaflet vein network of compound leaves, where the midrib in the leaflets of both species showed an embolism onset under a mean water potential of −0.91 ± 0.11MPa, while secondary and tertiary veins embolized later under water potentials of −1.47 ± 0.37 MPa.

A comparison of vulnerability thresholds measured using different techniques showed similar embolism vulnerability values between the optical technique and the air-injection method in stems and petioles for both species (Supplemental Table S1). Only the Pe estimations for the petioles of F. mandshurica diverged between the optical method and the air-injection method. We observed higher estimations for leaf Pe and P50 measured with the rehydration kinetics method in comparison with the optical visualization technique (Supplemental Table S1), although these differences could not be statistically tested due to a lack of replicate measurements using rehydration kinetics.

The hydraulic safety margins between Ψmd and P50 were significantly different across organs in both species (Figure 3). According to the results of the optical measurements on hydraulic vulnerability, safety margins (Ψmd–P50) in leaflets of both F. mandshurica and J. mandshurica were substantially smaller compared to the other organs of the shoot, with only 0.47 ± 0.05 MPa in F. mandshurica and 0.63 ± 0.06 MPa in J. mandshurica. Safety margins in stems were the largest in F. mandshurica (2.40 ± 0.09 MPa) and J. mandshurica (3.01 ± 0.089 MPa), while petioles show an intermediate level in hydraulic safety margin, with 1.21 ± 0.07 MPa in F. mandshurica and 1.85 ± 0.07 MPa in J. mandshurica (Figure 3, E and F). Similar differences across organs were observed when comparing a shorter safety margin, represented by the difference between Ψmd and Pe (Figure 3). In addition, safety margins calculated by the difference between Ψmd and Pe were negative (Figure 3, C and D) in the leaflets of F. mandshurica (−0.67 ± 0.07 MPa) and J. mandshurica (−0.71 ± 0.05 MPa), as well as in the petioles of F. mandshurica (Figure 3C). These negative safety margins based on Pe thresholds suggest that initiation of xylem embolism might have occurred in the leaflets of these two compound-leaved tree species during the peak of daily water stress in the dry season.

Figure 3.

Figure 3

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Mid-day water potentials and hydraulic safety margins in leaflets, petioles, and stems of F. mandshurica and J. mandshurica measured during a sunny day in mid-August. (A and B) Midday water potential (Ψmd), (C and D) the difference between Ψmd and air-entry water potential (Ψmd – Pe), and (E and F) the differences between Ψmd and xylem water potentials corresponding to 50% embolism accumulation (Ψmd – P50). Error bars show ± 1 se (n = 3). Different letters above or below the bars indicated significant differences between leaflet, petiole, and stem (one-way analyses of variance followed by LSD post hoc test, P < 0.05).

Discussion

Our study employed an optical method to quantify xylem embolism across all the organ types in the plant shoot (leaflet, petiole, stem) of two compound-leaved tree species. This standard approach provided deeper insights into vulnerability segmentation across the whole-shoot hydraulic system of trees with large compound leaves, clearly showing that leaflets and petioles are more vulnerable to drought-induced embolism than stems. Our results provide a potential physiological explanation for vulnerability segmentation mechanisms and adaptations of large sized compound-leaved tree species in forests that potentially experience marked periods of water deficits.

Embolism initiation and its propagation across leaflet, petiole, and stem

Multiple studies have shown that the optical technique produces similar estimates of embolism vulnerability compared to hydraulic methods (Brodribb et al., 2016b; Gauthey et al., 2020) providing high confidence that the embolized area quantified with the optical technique provided a good representation of vulnerability xylem function to water deficit-induced dysfunction. Using the optical technique, we confirmed that initial embolisms occur under relatively high water potentials in leaf veins and the leaf petiole, rendering the hydraulic system of the leaf at relatively high risk of drought-induced dysfunction (Nardini et al., 2001, 2003; Johnson et al., 2012; Brodribb et al., 2016a). The relatively high vulnerability of leaf veins can augment the decline in leaf water potential during drought stress and may function as a safety valve in protecting the more carbon costly stems (Brodribb and Holbrook, 2003, 2004; Liu et al., 2015). Comparisons between water potentials of embolism initiation (Pe) in different parts of the shoots in the two studied compound-leaved tree species suggest that embolism is much more prone to occur in the xylem of leaflets and petioles than in the stem xylem. Although measured mid-day water potentials in several species overlapped with the incipient cavitation predicted by the vulnerability measurements, it must be remembered that vulnerability measurements were made under nontranspiring conditions, while mid-day water potentials included a substantial pressure drop associated with cavitation. Assuming that there is a substantial pressure drop between the xylem and sites of evaporation (Scoffoni et al., 2016), it is likely that these leaves were still safe from cavitation at midday because xylem water potential is expected to be up to 1 MPa less negative than the venation. The substantially more negative Pe in stem xylem compared to mid-day stem xylem water potentials indicates that embolism is not routine in stem xylem, and that diurnal changes in stem hydraulic conductivity observed previously might be potentially attributed to measurement artifacts (Sperry, 2013; Wheeler et al., 2013; Trifilò et al., 2014; Lamarque et al., 2018).

Our continuous optical observations indicate that xylem embolisms propagate easily between connected leaf veins of the same order, and between neighboring vessels in the stem xylem but not between veins of different orders or from leaf petioles to stem xylem (Supplemental Movie S1–S3), which contributes to the maintenance of distinct embolism vulnerability between leaflets, petioles, and stems. Embolism spreading might occur as independent events in species with poor xylem network connectivity (Johnson et al., 2020). However, in species where vessel groupings are common, the synchronization of embolism usually observed between neighboring conduits indicates the importance of inter-conduit connections in mediating embolism propagation (Brodribb et al., 2017; Johnson et al., 2020). According to the air-seeding hypothesis, embolism propagates between adjacent xylem conduits through micropores in the pit membranes (Tyree and Sperry, 1989; Cochard et al., 1992; Tyree and Zimmermann, 2002; Schenk et al., 2015). The optical technique, employed here to study leaflets, petioles, and stems, directly captures the patterns of embolism propagation, and indicates the critical role of conduit connections to cavitation propagation.

In order for the vulnerability segmentation to be effective, mechanisms guaranteeing certain degrees of physical and/or physiological isolation between xylems of the leaflet, petiole, and stem should be crucial. Vessels are not completely randomly distributed across the plant vascular system, and they may end at the junction of different tissues or organs, such as stem–petiole transitions and petiole–leaflet major veins, or near branch nodes (André, 2005; Wolfe et al., 2016). Conduit end walls should be particularly concentrated at the junction of petioles and stems (Tyree and Ewers, 1991; Tyree and Zimmermann, 2002), making embolism propagation difficult between leaves and stems. Although this lack of conduit connections would decrease the efficiency of water flow from stem xylem to petiole xylem since the leaf insertion points usually have higher hydraulic resistance (Sperry et al., 2006), plants may invest more carbon (e.g. thicker cell walls) to more proximal xylem to make it more resistant to drought-induced xylem embolism by limiting the propagation of embolism basipetally from terminal parts of the shoot with lower water potentials (Guan et al., 2021). Furthermore, the water potential decrease from stem to petioles to leaf lamina contrasts with the variation of safety margins between them. The water potential drop generated as water traverses different organs is proportional to the hydraulic resistance of the organ in the hydraulic system. Since hydraulic resistance in leaflets accounts for >65% of the whole-shoot hydraulic system in both F. mandshurica and J. mandshurica (Song et al., 2018), large water potential gradient is limited to the leaflets of compound leaves. Indeed, substantially higher water potentials are observed in the stem xylem during plant active transpiration and, in parallel, stems present larger safety margins, minimizing their risks of embolism (Nolf et al., 2015; Johnson et al., 2018). Similarly, higher hydraulic vulnerability to embolism associated with greater degrees of vascular integration and greater xylem tension have been observed in more distal parts of large compound leaves (fronds) of fern species (Brodersen et al., 2012). Given that the magnitude of safety margins decreases from stems to petioles to leaflets in compound-leaved tree species, embolisms would first impact the more terminal parts of the shoot under unfavorable water conditions (Tyree and Ewers, 1991; Liu et al., 2015; Song et al., 2018).

The adaptation of vulnerability segmentation in shoots of compound-leaved trees

Compound leaf form is considered to represent an adaptation allowing rapid growth in tree species (Givnish 1978; Niinemets, 1998; Malhado et al., 2010). Consistently, compound-leaved tree species are found to have greater shoot hydraulic conductance than single-leaved tree species co-occurring in the same habitat and exhibit a risky stomatal control strategy allowing higher rates of photosynthetic gas exchange (Liu et al., 2015; Yang et al., 2019), which might bring potential high risks to the compound-leaved trees during drought events. The risky hydraulic strategy in the long petioles that function with small safety margins in both the studied compound-leaved tree species is apparently related to its low limitation to leaf photosynthesis during moderate water stress, such as under relatively high vapor pressure deficit during the midday (Salleo et al., 2000; Hao et al., 2011; Hao et al., 2013b; Liu et al., 2015). Under these conditions, a tight stomatal control should limit leaf transpiration and hence CO2 assimilation (Santiago et al., 2004; Zhang and Cao, 2009). However, even in relatively humid environments, unpredictable drought spells might occur. In these regards, having leaves and petioles that function like “hydraulic fuses” might be relevant for compound-leaved tree species. When facing these unpredictable severe drought events, although it represents a large carbon loss, dropping the whole leaf is apparently crucial to protect the integrity of xylem water transport in the upstream organs (Brodribb and Holbrook, 2003; Zufferey et al., 2011; Bucci et al., 2013; Pivovaroff et al., 2014; Liu et al., 2015; Song et al., 2018). The two species in the current study are limited to relatively humid habitats, growing along streams at low elevations with sporadic water shortages. During sufficient water supply conditions, high whole-shoot hydraulic conductance allows efficient water delivery to support high photosynthetic assimilation rates in these species with large compound leaves (Song et al., 2018; Yang et al., 2019). However, the habitat conditions of the studied species also imply the possibility of facing moderately dry conditions or even severe drought, for which a narrower safety margin in leaflets and petioles provides maximum hydraulic safety to protect the more carbon costly stems (Givnish, 1978, 1979).

The tradeoff between hydraulic efficiency and safety due to competing requirements for xylem structures have been reported in many studies, particularly in habitats with substantial water deficits (Martínez-Vilalta et al., 2002; Sperry et al., 2006; McCulloh et al., 2014), while the efficiency and safety conflict seems to be well balanced by the vulnerability segmentation of these trees with large compound leaves. Our results suggest that tree species with large compound leaves can preserve hydraulic efficiency when water is readily available and meanwhile guarantee hydraulic safety of the stem xylem under unpredictable severe drought stress by dropping their large compound leaves through HVS. Several works have found differences in drought sensitivity between different plant organs within an individual, using various methods to measure xylem embolism vulnerability (e.g. Alder et al., 1996; Sperry and Ikeda, 1997; Choat et al., 2005; Bucci et al., 2012; Scholz et al., 2014; Charrier et al., 2016). In contrast, other studies applying noninvasive methods have shown a lack of vulnerability segmentation between organs in woody and herbaceous species (Skelton et al., 2017; Lamarque et al., 2018). The application of standard noninvasive techniques such as the optical method, which can be simultaneously applied to different plant organs, on a wide diversity of plant species with various morphologies and ecological preferences would be useful in understanding the importance of the HVS as an adaptation to water stress.

Materials and methods

Study site and plant species

The study was carried out at the Research Station of Changbai Mountain Forest Ecosystem of the Chinese Academy of Sciences (128°28′E, 42°24′N; 736 m altitude), in the Jilin province, NE China. This region, as an important component of the world’s temperate forests, has a typical temperate continental climate, with a long and cold winter and a short and mild summer. A mixed forest of Korean pine (Pinus koraiensis) and broadleaf deciduous tree species is the most abundant forest type in this region, and this type of forest has a relatively high species richness among temperate forests (Cao et al., 2007). For the present study, we studied Manchurian ash (F. mandshurica Rupr. in the family Oleaceae) and Manchurian walnut (J. mandshurica Maxim. in the family Juglandaceae), which are important dominant species in the mentioned mixed forest of NE China (Sun et al., 2008; Yu et al., 2015). Both are light-demanding and fast-growing species favoring mesic habitats, with large compound leaves and long petioles (Song et al., 2018). All sampled plants used in this study were mature trees growing in the arboretum of the research station under similar environmental conditions, which minimizes potential phenotypic adjustments derived from growing conditions. Only sun-exposed shoots were sampled and measured. Both species have very long vessels with the mean vessel lengths measured using the air-injection method (Pan et al., 2015; Gao et al., 2019) being 104.1 ± 5.9 cm in F. mandshurica and 67.3 ± 3.1 cm in J. mandshurica, respectively. To minimize the effect of open vessels on the measurements, we sampled long shoots in both species with length of 132 ± 14 cm in F. mandshurica and 127 ± 12 cm in J. mandshurica, respectively. Embolism vulnerability in leaflets, petioles and stems was measured simultaneously on these long branches.

Embolism vulnerability measurements using the optical technique

During July–August 2017 (growing season), large branches from at least six healthy individuals for each of the two species were collected at predawn (4:00–5:00) in the botanical garden of Changbai Mountain. Branches were immediately placed in a water-filled bucket and transported to the laboratory with the top of the branch covered by at least two plastic bags with wet paper towels to prevent water loss. To avoid measurement inaccuracies, we only collected one branch at a time. Following the optical method protocol described by Brodribb et al. (2016b), we used three large branches from different individuals of each species to measure the hydraulic vulnerability in leaflets. To measure embolism vulnerability of the stems and petioles, we sampled another three large branches from the same three individuals during the same period of the growing season to measure hydraulic vulnerability based on the optical methods described by Brodribb et al. (2017). At the beginning of measurements, we maintained branches in a water-filled bucket and covered them with dark plastic bags for more than an hour to equilibrate the water potentials between leaflets, petioles, and stems. Upon removing the water supply, the vulnerability measurement of leaflets, petioles, and stems was carried out during the entire dehydration process. Embolism events were recorded using a flatbed scanner (Epson Perfection V800 Photo Color Scanner, Epson America, Inc.) combined with a stem psychrometer (PSY1, ICT International, Armidale, NSW, Australia) to record real-time water potentials of the branch in an air-conditioned room with temperature kept around 20°C. The sensor of the PSY1 psychrometer was installed on the stem of the middle part of the branch and the water potential measurements were recorded every 10 min. In order to prevent rapid dehydration of the branch and water potential imbalance between leaflets and adjacent stems, we kept the dark-adapted branch covered with a black plastic bag during the whole measurement period to keep the stomata closed and maintain a very slow dehydration rate. The water potential recorded by the PSY1 psychrometer overall showed a linear decrease over a relatively long time (24–30 h) during the experiment period (Figure 1).

Leaflet sample preparation and image capture

We chose a healthy and intact single leaflet near to the stem segment where stem xylem water potential was recorded using the PSY1 psychrometer. With the target leaflet still attached to the large branch during the whole process, we carefully placed it between two microscope slides on the flatbed scanner using adhesive tape (Figure 1B). The target leaflet was scanned in transmission mode, which allows an intensity of c. 40–80 mol quanta light to pass through the leaflet xylem to produce a transmitted light image. We scanned the target leaflet every 5 min for 3–4 d. Leaf xylem water potential was monitored simultaneously to estimate the water status of the branch. Since both species have very large leaves (leaf surface: 0.043 ± 0.004 m2 in F. mandshurica; 0.12 ± 0.004 m2 in J. mandshurica), water potentials were kept in balance between target leaflets and neighbor stems (variation between leaflet and adjacent stem was always less than 0.1 MPa). A stem psychrometer was connected to the adjacent stem xylem to provide a continuous measure of the water potential during dehydration. Stem psychrometers were connected to a small xylem region of the adjacent stems, which were prepared by carefully removing the bark. High-vacuum grease and parafilm were used to make sure the connection was under stable environmental conditions. Water potentials were continuously recorded every 10 min during the entire scanning process (Figure 1C).

Stem and petiole sample preparation and image capture

For the stems and petioles, we chose straight mature stems of c. 4–6 mm in diameter, only petioles bearing healthy leaflets were selected to carry out measurements. We carefully removed 20 mm of bark from one side of the target stem to expose the xylem using parallel axial cuts to avoid damage, and we verified that the xylem region was smooth, flat, and clean before scanning. For petioles of the two species, 6–10 mm xylem regions were prepared for scanning. Once the xylem region was exposed, we placed it on the flatbed scanner, and we firmly secured it with adhesive tape to fix the samples during the scanning process. Here, unlike the procedure used for leaflets, the target xylem region from either stems or petioles was scanned in reflective mode to capture images every 3 min during 3–5 d of dehydration (Figure 1B). A stem psychrometer was fitted as close as possible to the target xylem region being scanned in order to simultaneously record water potential (Figure 1C).

Image processing for embolism vulnerability measurements

After completion of the scanning process, we identified cavitation events by analyzing changes in the transmission of light through the leaflet xylem or in the reflection of stem and petiole xylem from image sequences. We used the image subtraction method in ImageJ (Schneider et al., 2012), which can highlight rapid changes in light transmission caused by air bubble expansion for leaflets and contrast produced by embolism for stems and petioles during the desiccation process. Changes in light transmission were filtered from those produced by the shrinkage movement caused by desiccation. We then used ImageJ to threshold differences throughout the image stack which could highlight the embolism events. We then used the “analyze-stack” function in ImageJ to automatically account each embolism event. Full details of the optical visualization method, including an overview of the technique, and scripts to guide image capture and analysis are available at http://www.opensourceov.org.

Quantifying the number of pixels per embolism event during desiccation allowed us to extract a time-resolved count of embolism events. We then estimated the percentage of total pixels cavitated, which produced a temporally and spatially resolved percentage of embolism dataset. We combined the time-resolved percentage embolism dataset with the psychrometer water potential timeline to estimate the leaflet, stem, and petiole xylem water potential thresholds associated with each embolism event (Figure 1D). Vulnerability curves are thus represented by the relationship between percentage cumulative embolisms and water potential. For consistency with other studies, we calculated the water potential responsible for 50% observed embolism (P50), which has been shown in several studies to be equivalent to the xylem water potential responsible for 50% loss of maximum conductivity (Brodribb et al., 2016b; Skelton et al., 2017; Gauthey et al., 2020). In addition, the air entry water potential (Pe), which is defined as the xylem water potential associated with 12% embolism events (Brodribb et al., 2016b; Skelton et al., 2018), was also estimated. The air entry water potential represents the critical point at which hydraulic functions begin to be damaged (Skelton et al., 2017). Mean P50 ± se (standard error) and Pe ± se (n = 3) for leaflets, petioles, and stems of each species were calculated.

Mid-day water potential measurements in different shoot organs

Water potentials were measured in sunny days during the growing season (mid-August 2017) using a pressure chamber (ZLZ-4, Lanzhou University, Lanzhou, China). For leaflets, mid-day water potentials (Ψmd) were measured at one time point under nonextreme water deficit conditions between 12:00 and 14:00 on six mature leaflets from sun-exposed branches of six different individuals for each species. Leaflet samples were cut from trees, sealed immediately in plastic bags containing moist paper towels, transported to the laboratory in a cooler, and measured within one hour after collection. For petioles and stems, six distal leaflets or the whole compound leaves from sun-exposed branches of different individuals were wrapped using aluminum foil and plastic bags containing moist paper towels in the evening before the measurement day to estimate petiole or stem water potential. In this way, water potentials in petioles and stems were assumed to be in equilibrium with these wrapped leaflets and whole compound leaves, respectively. Thus, we could measure the water potential of the wrapped leaflets and whole compound leaves to estimate the water potential of the petioles and branches connected to them.

Statistical analyses

Embolism vulnerability curves were obtained by fitting a sigmoid function model to the relationship between cumulative percentage embolism and water potential:

Percentage embolism=100−100/(1+ea(Ψ−b))

where a is a fitted parameter related to the slope of the curve and b is the Ψ associated with 50% embolism. One-way analyses of variance were used to test statistical significance in the differences of hydraulic-related traits between different organ types (i.e. leaflet, petiole, and stem), and mean comparisons were performed by least significant difference (LSD) post hoc test. Analyses were considered significant at P ≤ 0.05. Similar analyses were used to compare the hydraulic vulnerability values obtained with the optical visualization technique to those obtained using other methods. Hydraulic vulnerability data for the air-injection and rehydration kinetics methods were obtained from Liu et al. (2015). All statistical analyses were done with SPSS Statistics 19.0 (SPSS Inc., Chicago, IL, USA).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Table S1. Comparison of hydraulic vulnerability parameters calculated from vulnerability curves constructed using different methods in F. mandshurica and J. mandshurica trees growing in a common garden.

Supplemental Movie S1. The animated version of xylem embolism progression in a leaflet of Juglans mandshurica as shown in Figure 2.

Supplemental Movie S2. The animated version of xylem embolism progression in a petiole of Juglans mandshurica as shown in Figure 2.

Supplemental Movie S3. The animated version of xylem embolism progression in a stem of Juglans mandshurica as shown in Figure 2.

Supplementary Material

kiac034_Supplementary_Data
Click here for additional data file. (2.6MB, zip)

Acknowledgments

We thank the staff at the Research Station of Changbai Mountain Forest Ecosystems. We thank the members of Guang-You Hao’s lab for supporting this study.

Funding

This research was funded by the National Natural Science Foundation of China granted to G.Y.H. (31722013; 31870593) and J.S. (32101480), National Key R & D Program of China (2020YFA0608100), the Key Research Project from the Bureau of Frontier Science and Education Chinese Academy of Sciences (ZDBS-LY-DQC019), K. C. Wong Education Foundation (GJTD-201807), the Liaoning Revitalization Talents Program (XLYC1807204), Science and Technology Innovation Talent Program of Shenyang City (RC190143) and the ARC Centre of Excellence for Plant Success in nature and agriculture (CE200100015). S.T. received support from an IdEx-University of Bordeaux postdoctoral fellowship.

Conflict of interest statement. None declared.

Contributor Information

Jia Song, CAS Key Laboratory of Forest Ecology and Management & Key Laboratory of Terrestrial Ecosystem Carbon Neutrality Liaoning Province, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, Liaoning, China; School of Environmental and Geographical Science, Shanghai Normal University, Shanghai 200234, China; Yangtze River Delta National Observatory of Wetland Ecosystem, Shanghai Normal University, Shanghai 200234, China.

Santiago Trueba, University of Bordeaux, INRAE, BIOGECO, 33615 Pessac, France.

Xiao-Han Yin, CAS Key Laboratory of Forest Ecology and Management & Key Laboratory of Terrestrial Ecosystem Carbon Neutrality Liaoning Province, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, Liaoning, China.

Kun-Fang Cao, Plant Ecophysiology and Evolution Group, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, and College of Forestry, Guangxi University, Nanning, Guangxi 530004, China.

Timothy J Brodribb, Biological Sciences, School of Natural Sciences, University of Tasmania, Hobart, Tasmania 7001, Australia.

Guang-You Hao, CAS Key Laboratory of Forest Ecology and Management & Key Laboratory of Terrestrial Ecosystem Carbon Neutrality Liaoning Province, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, Liaoning, China.

 

G.Y.H. conceived the ideas; G.Y.H. and J.S. designed the study; J.S. and X.H.Y. conducted field and laboratory measurements; G.Y.H., J.S. and S.T. analyzed and interpreted the data; J.S., S.T. and G.Y.H. wrote the manuscript. K.F.C and T.J.B. contributed substantially to the revision of the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is Jia Song (jiasong@shnu.edu.cn).

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Supplementary Materials

kiac034_Supplementary_Data
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