| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
What's this? |
Ecology |
2Natural Science Division, Pepperdine University, Malibu, California 90263 USA; 3Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824 USA
Received for publication August 29, 2004. Accepted for publication April 7, 2005.
ABSTRACT
The response to freeze–thaw stress was examined for two co-occurring evergreen species, Malosma laurina and Rhus ovata. Laboratory and field experiments on adults and seedlings were made in the spring and winter in 1996 and again on adults in 2003 and 2004. Laboratory and field results indicated that the stem xylem for adults of M. laurina and R. ovata were similarly susceptible to freezing-induced cavitation (percentage loss of conductivity = 92 ± 2.6% for R. ovata and 90 ± 4.2% for M. laurina at 
–6°C). In contrast, leaves of M. laurina were more susceptible to freezing injury than leaves of R. ovata. Among seedlings in the field, leaves of M. laurina exhibited freezing injury at –4°C and total shoot mortality at –7.2°C, whereas co-occurring seedlings of R. ovata were uninjured. Surprisingly, R. ovata tolerates high levels of freezing-induced xylem embolism in the field, an apparently rare condition among evergreen plants. Rhus ovata avoids desiccation when xylem embolism is high by exhibiting low minimum leaf conductance compared to M. laurina. These results suggest a link between minimum leaf conductance and stem hydraulics as a mechanism permitting the persistence of an evergreen leaf habit in freezing environments.
Key Words: Anacardiaceae • California • cavitation • chaparral • embolism • Malosma laurina • Rhus ovata • xylem
Freezing survival of evergreen plants involves both the tolerance of the living tissues of the leaves and the function of the nonliving water transport system of the stems. For leaves or living cells, damage and subsequent death can occur when the water in living cells freezes or when cells dehydrate due to the extracellular freezing of water (Sakai and Larcher, 1987
). For stems, when sap in the xylem freezes, the gas dissolved in the sap comes out of solution because the solubility of gas is greatly reduced in ice compared to liquid sap. This can lead to the formation of emboli in xylem cells upon thawing if the bubbles do not go back into solution but instead expand in a process called cavitation (Yang and Tyree, 1992
). The likelihood that bubbles go back into solution when the sap thaws is greater for smaller bubbles and for plants with xylem sap under less tension (Yang and Tyree, 1992
; Langan et al., 1997
). In the absence of water stress, woody plants that have mean hydraulic vessel and tracheid diameters that are <35 µm are relatively resistant to cavitation caused by freeze– thaw stress, whereas stems that have hydraulic mean vessel and tracheid diameters >35 µm are susceptible to such cavitation (Davis et al., 1999
; Pittermann and Sperry, 2003
).
For an evergreen woody plant, if the xylem transport system is rendered permanently nonconductive due to cavitation caused by freezing, then tolerance of the leaves to freezing becomes moot because the leaves will desiccate without an adequate supply of water. Because of this, the stem xylem should be greater than or equal in its resistance to cavitation caused by freeze–thaw stress compared to the degree of freeze–thaw stress damaging leaves. Consistent with this model, the leaves of the chaparral shrub Ceanothus crassifolius only tolerate freezing temperatures to –18°C, whereas its stems resist severe freeze–thaw embolism when frozen to –25°C at a branchlet water potential (
x) of –5 MPa (Ewers et al., 2003
). Further, both the leaves and stems of the chaparral shrubs C. megarcarpus and C. spinosus are intolerant of a –10°C freeze–thaw, i.e., the leaves become damaged and the stems become highly embolized (95–100% loss in hydraulic conductivity) when frozen to –10°C and subsequently thawed at a x of –5 MPa (Langan et al., 1997
; Boorse et al., 1998a
; Ewers et al., 2003
).
A broad-leaved evergreen plant that does not appear to fit these patterns for chaparral species is Rhus ovata S. Watson. This plant commonly occurs in cold-air drainage basins and at higher elevations in the chaparral where subfreezing temperatures are quite common, yet it has relatively large diameter vessels. Our preliminary results indicated that it had vessels as large as 80 µm in diameter. These large vessel diameters are similar to another evergreen shrub, Malosma laurina (Nutt.) Brewer and S. Watson, also of the Anacardiaceae (mean vessel diameter = 59.2 µm; Carlquist and Hoekman, 1985
). By comparison, Carlquist and Hoekman (1985)
found a mean vessel diameter of 29.2 µm, for 41 chaparral species.
Rhus ovata and M. laurina share many similarities. For example, they broadly overlap in their distributions in southern California, are evergreen, resprout and recruit from seed after wildfire (facultative sprouting), have similar leaf size and shape, have similar minimum seasonal water potentials (Miller and Poole, 1979
), and have similar resistance to cavitation caused by water stress (S. Davis, unpublished data).
However, M. laurina and R. ovata are not similar in their freezing tolerance. Malosma laurina is highly vulnerable to freeze–thaw embolism and is restricted to nonfreezing sites in the chaparral and coastal sage scrub of southern California (Langan et al., 1997
). Rhus ovata shows elevated native embolism at freezing sites, yet it persists with no visible damage to its leaves or stems at sites where winter low temperatures can reach –12°C (Langan et al., 1997
). This presents an interesting paradox because stems of R. ovata are highly vulnerable to cavitation caused by freeze–thaw stress, whereas its leaves apparently tolerate freezing temperatures and do not desiccate when the stems are highly embolized.
Here we examined the response to freeze–thaw stress for two chaparral species, M. laurina and R. ovata, that have overlapping distributions in southern California. We hypothesized that, for R. ovata, the stems would experience high levels of embolism following freeze–thaw, whereas the leaves would not suffer damage. In contrast, for M. laurina, we predicted similar susceptibility to freeze–thaw stress for both stems and leaves. Some have suggested that the parenchyma of stems may be killed by freezing temperatures and that this can lead to cavitation (Pockman and Sperry, 1997
; Martinez-Vilalta and Pockman, 2002
; Pittermann and Sperry, 2003
). To address this possibility, we examined death among ray parenchyma cells following freezing treatments to assess if cavitation caused by freeze–thaw was concurrent with the death of xylem parenchyma. In addition, we examined traits that would allow R. ovata to avoid or tolerate cavitation caused by freezing, such as phenology of leaf production and reduced leaf conductance to water vapor. Finally, we present unique data from a natural field experiment causally linking freezing temperatures to differential seedling mortality in R. ovata and M. laurina growing side by side.
MATERIALS AND METHODS
Plants and study sites
We studied adults and seedlings of two co-occurring chaparral species, Malosma laurina (formerly Rhus laurina) and Rhus ovata (nomenclature follows Hickman, 1993
).
We studied at two sites: a warm site near the ocean and a cold site 6 km inland from the coast near Cold Creek Canyon (described in Langan et al., 1997
and Ewers et al., 2003
). The lowest temperature reported at the cold site is about –12°C, and temperatures regularly reach –8°C for a seasonal low (Ewers et al., 2003
). We monitored temperature during the study with a max–min thermometer (McMaster Carr, Los Angeles, California, USA), and previous studies have systematically documented temperatures throughout the site (Langan et al., 1997
). The warm site was located in the city of Malibu near Pepperdine University. This site served as a nonfreezing control site for experiments with M. laurina seedlings. The minimum temperature at this site rarely reaches 0°C due to its proximity to the Pacific Ocean. We monitored temperature and rainfall at this site with a max–min thermometer and manual rain gauge (All Weather Rain Gauge, Productive Alternative Inc., Fergus Falls, Minnesota, USA).
At our cold study site, adults of M. laurina and R. ovata are differentially distributed, whereas seedlings of the two species can co-occur following wildfire. At this site, adults of M. laurina are restricted to ridge crests that remain 3–4°C warmer than valley bottoms where cold air drainage is common (Langan et al., 1997
; Ewers et al., 2003
). Severe freezing events every 10 to 20 yr can cause total shoot dieback of M. laurina adults (–11°C in 1990; Boorse et al., 1998b
; Fig. 2A), yet M. laurina can persist along the ridge crests at the site by resprouting from a large root crown (DeSouza et al., 1986
; Frazer and Davis, 1988
; Thomas and Davis, 1989
). Rhus ovata occurs throughout the site and overlaps in its distribution with M. laurina along the ridge crests. Adults of M. laurina were never observed in the cold-air drainages of valley bottoms, but seedlings emerged in the months following a wildfire on 21 October 1996 and persisted throughout the summer and fall of 1997. Presumably, the source of the seedlings was the adults along the ridge crests, and their germination was promoted by the wildfire. Rhus ovata seedlings also germinated after the wildfire in the valley bottoms where they co-occurred with M. laurina seedlings.
|
Response of branches and leaves to freezing and thawing
For adults, response to freezing temperatures was evaluated by freezing branches in a cooling chamber (described in Langan et al., 1997
) in May and June 1996 (springtime) and again in January and February 2003 (wintertime). Cooling rate was controlled at –0.08°C/min between 15 and 0°C and a rate of –0.02°C/min between 0 and –10°C. Warming between –10 and 15°C occurred at a rate of 0.08°C/min. These cooling and thawing rates were chosen because they matched the rates measured on leaves and stems of M. laurina and R. ovata in the field at our cold study site (Langan et al., 1997
).
For treatment in the cooling chamber, large branches (
1 m in length) were removed from plants in the field at predawn, double-bagged, and brought back to the laboratory in 30 min. In the laboratory, excised branches were recut under water and left to sit with their excised ends submerged in water for about 2 h. Before and after rehydration, branchlet water potential (x) was estimated on two to four terminal branches using a pressure chamber (PMS, Corvalis, Oregon, USA). After hydration, a branch was inserted into the cooling chamber and a freeze–thaw cycle was begun. Temperature of the xylem was estimated by inserting a thermocouple just under the bark on a side branch, and as a control, a thermocouple was attached to a dowel rod. A second branch was bagged and left in an air-conditioned laboratory for the duration of the freeze–thaw treatment, serving as an unfrozen control. Both the control and frozen branches were kept hydrated by maintaining their excised ends in a bucket of water throughout the experiment. After the freeze– thaw treatment, x was again estimated for the treated and control branches.
The amount of embolism caused by the freeze–thaw treatment was estimated as the percentage loss of hydraulic conductivity (PLC) following the freeze–thaw in comparison to the unfrozen control. Following the freeze– thaw, no less than three lateral stems (about 14 cm long) were removed from both the treated and control branches by cutting them under water. For these stems, hydraulic conductivity (Kh; kg · m · MPa–1 · s–1) was measured by perfusing ultrafiltered (0.1 µm pore filter) de-gassed acid solution through them at low pressure (
2 kPa; Sperry et al., 1988
; Stiller and Sperry, 2001
). A low pH solution (pH 2, HCl) was used to prevent microbial growth (Sperry et al., 1988
). Following initial measurement of Kh, stems were flushed with acid solution at about 100 kPa for 60 min. The post-flushing Kh represented maximum Kh (Khmax), i.e., Kh with no emboli in the xylem. Percentage loss of hydraulic conductivity (PLC) was calculated as (1 – Kh/Khmax) x 100.
Freezing damage to living cells of leaves and stems was assessed. For leaves, damage was assessed using dark-adapted fluorescence (Fv/Fm) measured before and after the freeze–thaw treatments. Chlorophyll fluorescence from photosystem II is a fast and reliable indicator of low temperature stress of chloroplasts (Larcher, 1995
; Loik and Redar, 2003
; Percival and Henderson, 2003
). In addition, previous work has found that estimating leaf damage following freezing using Fv/Fm for M. laurina and R. ovata gave similar results to three other methods including electrolyte leakage (Boorse et al., 1998b
). Following the freeze–thaw, cuvettes were attached to six leaves of the treated and control branches to dark-adapt them for 20 min before Fv/Fm was measured using a pulse-modulated fluorometer (Opti-Sciences, OS1-FL, Tyngsboro, Massachusetts, USA).
For stems, the vitality of parenchyma cells was estimated using the fluorescent stain fluorescein diacetate (Rotman and Papermaster, 1966
; Windholm, 1972
; Boorse et al., 1998b
). In a viable cell, the relatively nonpolar and nonfluorescent fluorescein diacetate diffuses across the cell membrane where it is hydrolyzed by esterases into the more polar and fluorescent fluorescein. If the cell membrane is intact and the esterases are functional, the fluorescein will accumulate in the cell faster than it leaks out and fluoresce green when excited by blue actinic light (Rotman and Papermaster, 1966). If the esterases are not functional or the membrane is damaged then the fluorescein will not form and accumulate in the cell, and florescence will not occur.
Control and freeze–thaw stems were thin-sectioned and placed in 10 mL of deionized water to which 0.1 mL of 0.01% fluorescein diacetate in acetone was added. After 1 min, another 10 mL of deionized water was added to the solution. Sections were incubated in the solution for about 10 min for R. ovata and 60 min for M. laurina to allow the dye to infiltrate the cells. In preliminary trials, the dye infiltrated more slowly into M. laurina than into cells of R. ovata, necessitating a longer incubation time for M. laurina. After the incubation, sections were mounted in deionized water and viewed under blue fluorescent light (excitation filter BG-12) using an epifluorescence microscope (Nikon, Garden City, New York, USA) at 200x magnification. Control stems were sectioned and stained the same as treated stems. In addition, a second control stem was submerged in liquid nitrogen for about 10 s, thawed at room temperature, and stained. This second control verified that fluorescence was not present when cells were dead.
The injury of leaves in response to cooling temperatures was estimated in three ways during March and April 1996. Branches were frozen in a cooling chamber (as described earlier) to increasingly negative temperatures. For every 1°C decrease in temperature below 0°C, leaves were carefully removed from branches with clippers attached to a meter stick so as to not alter the temperature in the cooling chamber. Leaves were immediately placed in a sealed container in an ice chest to facilitate gradual thawing overnight. The lethal temperature at 50% cell death or damage (LT50) was estimated using Fv/Fm, vital staining with fluorescein diacetate, and a color change score using the Munsel color chart (1977 ed., Forestry Supply, Jackson, Mississippi, USA). All of the techniques to estimate the LT50 for leaves generally agreed within 1°C and are described in detail in Boorse et al. (1998b)
.
The effect of freezing on stem conductivity for R. ovata was also estimated on 21 January 2004 by measuring PLC for plants growing in the field following four subzero temperature events of –6°C in December 2003, with the lowest being –7.5°C on 19 December 2003. For these measurements, stems were removed from seven plants. All cuts to stems were made under water to prevent air from entering the xylem. Once removed, stems were double bagged and transported to the laboratory in <60 min. In the laboratory, stems were recut under water to 10 cm. The PLC was measured as described and represents an estimate of native-state embolism.
Experiments with seedlings
We sampled seedlings of M. laurina and R. ovata that germinated after a wildfire on 21 October 1996. Following this fire, the cold site was surveyed, and all M. laurina seedlings were tagged, mapped, and followed over time. All seedlings at our cold study site were found in the cold valley bottom. Apparently, the seedlings that germinated after the wildfire did so after the coldest part of the winter, which is typical for M. laurina. That is, M. laurina typically germinates in January and February (Thomas and Davis, 1989
), and December is often the month with the minimum temperature for the season at our cold study site (Langan et al., 1997
). About 7% of the M. laurina seedlings survived the subsequent dry season in 1997, and these are the seedlings we sampled in December 1997. At this time, we located co-occurring R. ovata seedlings for comparison. We measured Fv/Fm and survivorship for 12 seedlings of M. laurina and R. ovata following freezing and subfreezing temperatures. In addition to the 12 M. laurina seedlings at the cold site, as a control, Fv/Fm was measured for 10 seedlings of M. laurina at a site about 5 km away that did not have freezing temperatures because it is near the Pacific Ocean. Seedlings from the warm and cold site were approximately the same age because they all germinated after the Calabasas/Malibu wildfire of 1996.
Leaf temperature of seedlings at the cold and warm sites was monitored continuously in December 1997. Abaxial leaf temperature was measured with a thermistor and datalogger (Onset, Pocasset, Massachusetts, USA).
Vessel diameter
Vessel diameters were measured for stem segments exposed to freezing temperatures in the cooling chamber. Transverse sections were made at the midpoint of 6 mm diameter stem segments with a microtome (American Optical Company, Buffalo, New York, USA), and vessel diameters were measured in four nonoverlapping areas (two near the pith and two near the stem surface), using a compound microscope (Nikon) with a digital camera (Spot RT Color Camera, Diagnostic Instruments Inc., Sterling Heights, Michigan, USA) and imaging software (ScionImage version Beta 3b, Scion Corporation, Frederick, Maryland, USA). The number of vessels measured per cross section ranged from 65 to 155, and a pooled mean was calculated for each species. We also expressed vessel diameters in terms of mean hydraulic vessel diameter, 
d5/d4, where d = vessel diameter (Sperry et al., 1994
).
Minimum leaf conductance in the laboratory
We hypothesized that to tolerate high levels of stem embolism in a field environment after freeze–thaw stress, R. ovata would have a lower minimum leaf conductance compared to M. laurina. Transpiration and whole leaf conductance (stomatal and cuticular) were assessed for excised leaves of both species in June and July 2003 and February 2004. Because results from each of the three sampling dates were similar, data are only presented from July 2003 and February 2004. Six branches (1 m in length) were removed from plants at our cold study site in Cold Creek Canyon, immediately bagged and returned to the laboratory in 30 min. Stems were then recut under water and left to sit with their excised ends submersed in water for about 12 h. After hydration, inside a humid chamber, two mature leaves were removed from each branch and labeled, and cut surfaces of petioles were covered with fast-drying epoxy (Quick Set Epoxy, Loctite, Rocky Hill, Connecticut, USA) to prevent water loss from cut surfaces. Upon removing leaves from the humid chamber, their mass was measured, with a 0.01 mg resolution analytical balance (AE163, Mettler-Toledo, Columbus, Ohio, USA), as was dark-adapted Fv/Fm. Leaves were then placed on a mesh screen on a rack suspended about 10 cm above a laboratory bench top in June and July and in a controlled environment chamber in February. Light intensity was low: photosynthetically active radiation level for experiments in June and July 2003 was 12 µmol · m–2 · s–1 and in February 2004 it was at 55 µmol · m–2 · s–1. In the June, July, and February experiments, the vapor pressure deficit ranges were 1.4–2.3, 0.5–1.2, and 1.2–1.9, respectively, and the air temperature ranges were 21–22°C, 20–25°C, and 21– 22°C, respectively. The mass and Fv/Fm were measured 30 min after the initial measurement and for every hour after that for the first 5 h. Measurements were then made every 1 to 8 h until the mass remained constant between measurements and Fv/Fm was not different from 0. The dew-point temperature (Td) (Hygrometer, Edge Tech, Milford, Massachusetts, USA) and the air temperature adjacent to leaves (Ta) were measured each time the leaf mass was measured. Vapor pressure deficit (VPD) between the leaves and the air was calculated as esat(Td)–esat(Ta), where esat is the saturation vapor pressure (kPa) (Campbell and Norman, 1998
). Measurements indicated that the leaf temperature was not different from the air temperature. Whole leaf conductance (gleaf; mmol · m–2 · s–1) was calculated from the following relationship:
 | (1) |
) and Pa is atmospheric pressure (102.28 kPa in Malibu, California). The leaf area was multiplied by two because water was being lost from both leaf surfaces, a common practice when estimating gleaf (Kerstiens, 1996
). The area of the leaves was measured with a leaf area meter (Li-3100, Li-Cor Inc., Lincoln, Nebraska, USA).
Diurnal measurements in a field environment
To determine if R. ovata had low gs and was under water stress resulting from high levels of embolism, diurnal gs and x were measured on R. ovata in the valley bottom and on M. laurina along the ridge crest immediately upslope from R. ovata at our cold study site on 6 February 2004. Stomatal conductance was measured with a porometer (Li-1600, Li Cor Inc.). Water potential was measured with a pressure chamber on branchlets in the field (PMS). Both gs and x were measured every 2 or 3 h from predawn hours until dusk with one exception; we could not measure gs at predawn because the temperature was below freezing, preventing porometer function within manufacturer's specifications.
Tissue water relations
To assess if osmotic adjustments or tissue capacitance could explain the differential freezing tolerance between R. ovata and M. laurina, we estimated the capacitance and tissue water relations for leaves of both species using a pressure-volume technique modified from Tyree and Hammel (1972)
. Twelve branches, about 25 cm long, from six individual plants of each species were cut under water at 1800 hours on 12 May 2004. The cut end of each branch was immediately submerged in water, bagged, and allowed to rehydrate for 12 h for R. ovata and 24 h for M. laurina. "Plateau effects" described by Kubiske and Abrams (1991)
, indicating the filling of airspaces in leaves during saturation, were not apparent. Parameters for tissue water relations were analyzed and calculated following Koide et al. (1989)
.
Statistics
Data were analyzed using t tests and ANOVAs (Minitab v.14.12, Minitab, Inc., State College, Pennsylvania, USA), and transformed as necessary to satisfy assumptions of statistical models. When comparing treatments, differences were considered significantly different at alpha 0.05. If P was 0.05, we reported comparisons as similar or not different.
RESULTS
Percentage loss of hydraulic conductivity (PLC) of hydrated stems did not differ between R. ovata and M. laurina when exposed to freeze–thaw stress. Among individuals sampled in May and June 1996, PLC for stems frozen to –6°C was greater than 92% for both species, and greater than that for control stems (data not shown). Branchlet water potentials (x) for stems frozen to –6°C were –0.66 MPa for M. laurina and –0.55 MPa for R. ovata. The x for control and frozen stems were not different. Additional experiments performed in January and February 2003 again showed that the xylem of M. laurina and R. ovata exhibited similar PLC when exposed to freezing temperatures (Fig. 1A, B). For both species, PLC for stems frozen to –10°C was greater than that of unfrozen control stems. Branchlet water potential for treated stems was not different from that for control stems for both genotypes: x = –0.70 MPa for M. laurina and –0.97 MPa for R. ovata. To verify laboratory experiments with R. ovata, native-state PLC and leaf-specific conductivity (Kl) were measured on 21 January 2004 following multiple freeze–thaw events in December 2003 and January 2004. At this time, PLC and Kl were 92 ± 1.9% and 0.45 ± 0.16 x 10–4 kg · s–1 · MPa–1 · m–1, respectively, and predawn x was –1.88 ± 0.09 MPa. For M. laurina along the ridge crests on 3 March 2004, PLC was 70 ± 5.0% and Kl was 2.37 ± 0.35 x 10–4 kg · s–1 · MPa–1 · m–1.
|
0) compared to unfrozen control leaves, whereas for R. ovata, the Fv/Fm for frozen and unfrozen leaves did not differ (Fig. 1C, D). The ray parenchyma of stems for both species appeared to survive freezing temperatures of –10°C (Fig. 2C, G). Vitally stained stem parenchyma cells exposed to –10°C fluoresced similarly to cells of unfrozen control stems (Fig. 2C, D, G, H). In other words, freezing to –10°C caused no detectable cell death in stems of either species. The –80°C control did not fluoresce, indicating that our fluorescein diacetate stain was not causing fluorescence in nonliving cells (Fig. 2B and F).
The vessel diameters for M. laurina and R. ovata were not different (Fig. 3). For example, the mean, the hydraulic mean, and the maximum vessel diameters for M. laurina (27.4 ± 3.2, 46.8 ± 6.0, and 64.2 ± 7.3 µm, respectively) were not different from those of R. ovata (25.9 ± 1.4, 40.3 ± 2.6, and 55.4 ± 3.2 µm, respectively).
|
|
x are also shown during the dry-down experiment (Fig. 5D, I, E, J, respectively). These values were estimated from pressure-volume curves of leaves for the two species. After 24 h, for experiments in July and February, the gleaf for the two species did not differ, but by this time M. laurina leaves appeared to be depleted of water, based on the leveling of the water loss per unit leaf area and the RWC.
|
|
x for M. laurina and R. ovata measured at our cold study site on 6 February 2004 showed that gs and x were greater for M. laurina compared to R. ovata (Fig. 6A, B). Branchlet water potential was more than two times lower for R. ovata compared to M. laurina for all hours of the day (Fig. 6A). Stomatal conductance for R. ovata was about half that for M. laurina at 0800, 1515, and 1700 hr (Fig. 6B). At 1015 and 1315 hr, gs was not different between the two species (Fig. 6B). The day was clear and sunny, with photosynthetically active radiation peaking at 1200 hours at 1500 µmol · m–2 · s–1 (Fig. 6C). Low temperature overnight (between 5 and 6 February) was –4°C, and the high temperature on 6 February was 23.6°C (data not shown).
|
x and the timing of new leaf growth were different between M. laurina and R. ovata at our cold study site in the winter months of 2004 (Fig. 7A, B). Branchlet water potentials measured at predawn (pd) were lower in February, March, and April 2004 for R. ovata compared to M. laurina (Fig. 7A). In June (Julian days 153–182), pd for R. ovata and M. laurina converged and were no longer different (Fig. 7A). The timing of growth for new leaves occurred about two months later in the season for R. ovata compared to M. laurina (Fig. 7B). Some plants of M. laurina initiated new leaves in early March (Julian day 70), whereas R. ovata did not begin growth until early May (Julian day 130) (Fig. 7B). All plants of M. laurina initiated new leaves by early April and by late May for R. ovata (Fig. 7B). To put these measurements into context, December, January, and February were the coldest months, with at least 10 nights with lows of –4°C or lower, with a minimum of –7.5°C on 19 December 2003 (Fig. 7C; data for December is not shown). No subzero temperatures were recorded after 24 April 2004 (Julian day 115). Precipitation from January through March 2004 is shown in Fig. 7D.
|
The freezing results for R. ovata present an interesting paradox. As predicted from their large vessel diameters, stems for R. ovata and M. laurina experienced over 90% loss in hydraulic conductivity at temperatures of 6°C. At first glance, this result would seem paradoxical for R. ovata, because it is evergreen and presumably the leaves will desiccate if the xylem does not supply them with water year round. For example, a co-occurring evergreen chaparral species, Ceanothus crassifolius, undergoes dieback of side branches when stems are highly embolized (Davis et al., 2002
). Malosma laurina similarly had more than a 90% loss in hydraulic conductivity, but leaf desiccation is not a factor because freezing temperatures of –6°C caused both leaf and stem death for this species (Fig. 2A; Langan et al., 1997
). Besides our cold site, R. ovata occurs at inland sites in California and Arizona at elevations up to 1300 m (Hickman, 1993
), where frosts of –6°C are common in the winter. Maintaining water flow to leaves may be especially important during winter months in southern California, where a night of subzero temperatures may be followed by a warm, sunny day. We conclude that R. ovata can tolerate high levels of embolism while maintaining high leaf area (Fig. 2E), a combination that is apparently rare among evergreen woody plants, based on available data (cf. Sperry and Sullivan, 1992
; Sperry et al., 1994
; Lipp and Nilsen, 1997
; Sobrado, 1997
; Sparks and Black, 2001
; Cavender-Bares and Holbrook, 2001
; Cordero and Nilsen, 2002
; Mayr et al., 2003
).
Percentage loss of conductivity is a relative measure of embolism and may not be the clearest measure of how adequately leaves are supplied with water from stems compared to the more direct measure of leaf specific conductivity (Kl) (Tyree and Ewers, 1991
). On 21 January 2004, following multiple freeze–thaw events in December and January, Kl for stems of R. ovata was 0.45 ± 0.16 x 10–4 kg · s–1 · MPa–1 m–1, which is low among woody angiosperms (Stratton et al., 2000
; Cavender-Bares and Holbrook, 2001
). At this time, stems displayed a 92% loss in conductivity. In comparison to R. ovata, stems for M. laurina around this time (3 March 2004) were less embolized (70% loss of conductivity for M. laurina) and Kl for M. laurina was more than five times higher at 2.37 ± 0.42 x 10–4 kg · s–1 · MPa–1 · m–1. The high PLC and low Kl for R. ovata indicate that its stems were highly embolized, and water supply to its leaves was greatly reduced during winter months.
Although we focused on stems, the location of minimum conductance to water flow in plants may also be located in the roots or leaves. We did not measure the xylem conductance of roots or leaves, but we think that stem conductance is likely a chief resistance to water flow following freeze–thaw stress at our study site. This is because it is very unlikely that the roots experienced freeze–thaw stress because they are insulated by the surrounding soil. For leaves, vessel diameters are typically smaller than those in stems, which would theoretically reduce the severity of embolism following freeze–thaw stress (Davis et al., 1999
; Pittermann and Sperry, 2003
).
A reduction of water supply to leaves from embolism would be expected to cause a decline in x or stomatal conductance (gs), and we found evidence for both. The lowest published values for x at midday for R. ovata are about –3.0 MPa (Poole and Miller, 1981
; Vankat, 1989
). Branchlet x at midday on 6 February 2004 for R. ovata in our study was –3.4 MPa, which is lower than any previously published values for this species indicating that plants were under considerable water stress. In addition to low x, R. ovata also exhibited low gs. For field measurements on 6 February 2004, the maximum and minimum gs for leaves of R. ovata (gs = 31 and 20 mmol · m–2 · s–1, respectively) were both about half the values measured for M. laurina (gs = 67 and 37 mmol · m–2 · s–1, respectively). These gs values for R. ovata are extremely low compared to other woody plant species (cf. Körner, 1995
), including adult chaparral species during summer drought (Davis and Mooney, 1985
; Thomas and Davis, 1989
).
The high degree of water stress for R. ovata, manifested as low x and gs, in February 2004 was probably not due to lack of soil moisture. The predawn and midday x values for M. laurina (–0.80 and –1.69 MPa, respectively) on 6 February 2004 indicate relatively high soil moisture. January and February are typically among the wettest months in southern California, and there were multiple rainfall events in January and February 2004. Instead of lack of soil moisture, the low x and gs for R. ovata are likely due to high embolism and low Kl of stems, resulting in a reduced supply of water to its large canopy of evergreen leaves.
Cavitation caused by freezing and thawing may be a purely physical process controlled by x and the diameter of xylem vessels. Alternatively, death of xylem parenchyma cells may contribute to losses in hydraulic conductivity of stems when they are frozen and thawed (Pockman and Sperry, 1997
; Pittermann and Sperry, 2003
). Davis et al. (1999)
found that woody plants with hydraulic mean vessel diameters less than 35 µm at x = –0.5 MPa show high resistance to cavitation caused by freeze–thaw treatments, and that plants with hydraulic mean vessel diameter greater than 35 µm are susceptible to such cavitation. Their work was confirmed by Pittermann and Sperry (2003),
who found a similar critical conduit diameter for freezing susceptibility among species that have tracheids. These two studies support a physical model of freezing-induced xylem cavitation that is largely controlled by vessel or tracheid diameter. Parenchyma cells may also be involved in cavitation caused by freezing. Some plants (Larrea tridentata and Ginkgo biloba) show a negative correlation between freezing temperature and PLC (Pockman and Sperry, 1997
; Pittermann and Sperry, 2003
). This has been interpreted as an indication that ray parenchyma cells are progressively killed by decreasing temperatures, and this results in increasing PLC.
Our results support a physical model of cavitation caused by freezing for two reasons. First, both M. laurina and R. ovata were highly susceptible to cavitation caused by freezing, and their hydraulic mean xylem vessel diameters were greater than 35 µm. Second, we used a vital stain to assess if xylem parenchyma were killed by freezing temperatures of –10°C, and our results indicate that the parenchyma survived these freezing temperatures for both species. Because the parenchyma survived and the xylem had high PLC, we conclude that the PLC resulted primarily from the large vessel diameters and was not due to mortality of parenchyma cells. To our knowledge, these results are the first to directly examine PLC and survivorship of parenchyma for frozen stems. We do not rule out a role for parenchyma death in conductivity losses for other species, and it would be useful to assess viability of cells for Larrea tridentata and Ginkgo biloba to verify cell mortality of frozen stems.
Although the xylem parenchyma cells survived the –10°C treatment in the laboratory, such a freeze would kill shoots of M. laurina growing in a field environment (Fig. 2A; Langan et al., 1997
). This discrepancy seems to suggest that the death of the parenchyma cells in stem-xylem following freeze–thaw stress is not immediate and that their mortality may not be directly due to freeze–thaw stress. For example, in the field, embolism caused by freeze–thaw stress may lead to the desiccation and ultimately the death of xylem parenchyma cells. Alternatively, freeze–thaw stress may injure the cells and trigger programmed cell death (apopotosis), and not until sometime later are they killed (Arora and Palta, 1988
).
The leaves for R. ovata are clearly important for its ability to tolerate freezing temperatures in the field and for explaining its greater tolerance of freezing temperatures than M. laurina. Leaves for R. ovata adults and seedlings were not measurably damaged by freezing treatments of –10°C in the laboratory and –7.2°C in the field, respectively. In contrast, leaves for adults of M. laurina suffered damage at –6°C and seedlings experienced 100% mortality at –7.2°C. Freezing tolerance for R. ovata is better explained by the ability of its leaves to tolerate freezing temperatures and reduced water availability than the ability of its stems to resist cavitation caused by freezing. For M. laurina, cavitation of stems and damage to leaves both occurred at about –6°C, indicating that both are important in explaining the lack of freezing tolerance for M. laurina.
When extracellular ice forms it grows by withdrawing water from living cells, causing them to dehydrate (Ashworth and Pearce, 2002
). Therefore, the greater tolerance of subzero temperatures for leaves of R. ovata may be related to their tolerance of lower x than leaves of M. laurina. This appears to be the case as leaf x for R. ovata declined to –5 MPa, while dark-adapted Fv/Fm remained 0.8 (Fig. 5C, E, H, J). By comparison, leaf x values of –4.5 MPa for M. laurina lead to a decline in dark-adapted Fv/Fm values below 0.8 (Fig. 5C, E, H, J).
Another important feature of R. ovata leaves is that they are better able to reduce their conductance to water vapor than leaves of M. laurina. This was found among plants growing in the field and for experiments using excised leaves in the laboratory. For field measurements on 6 February 2004, the maximum and minimum gs for leaves of R. ovata (gs = 31 and 20 mmol · m–2 · s–1, respectively) were about half those for co-occurring M. laurina (gs = 67 and 37 mmol · m–2 · s–1, respectively). Laboratory experiments using excised leaves to estimate minimum whole leaf conductance (stomatal and cuticular conductance; gleaf) had similar results: the minimum gleaf for R. ovata was about half that of M. laurina. If stems for R. ovata become highly embolized following a freeze–thaw event and the supply of water to the leaves is reduced, it becomes imperative that the leaves reduce their demand for water. Apparently R. ovata is capable of making this leaf-level adjustment. It is unclear if the difference in minimum gleaf between R. ovata and M. laurina is due to differences in minimum stomatal conductance or differences in cuticular conductance.
The phenology of R. ovata may also enable it to grow in environments prone to winter freezing. Rhus ovata did not develop new leaves until 12 May 2004, which was about 2 months later than M. laurina. Delayed growth is an adaptation for some large-vesseled species, e.g., Quercus species, to avoid freezing-induced cavitation in the late spring (Sperry et al., 1994
). At our field site, there are two oak species with large vessel diameters, Q. agrifolia and Q. berberidifolia, both of which leafed out about a month earlier than R. ovata (Pratt, personal observation). Delayed spring growth for R. ovata may be an adaptation to avoid late freezing events that might cause cavitation and disrupt growth in the spring and summer. However, a more likely explanation is that the high winter embolism and low predawn x values for R. ovata prevented the necessary turgor pressure for cell elongation and leaf expansion.
Recovery from winter embolism for R. ovata probably does not involve refilling of previously embolized vessels because low x values likely preclude refilling during the winter months. In addition, at our cold study site, native embolism for R. ovata has been sampled on more than 20 separate occasions and has never exhibited native PLC < 40% (S. Davis, unpublished data) suggesting that, if embolism refilling occurs, it is incomplete. We suspect that R. ovata largely depends on new xylem growth to supply dividing cells and developing leaves with adequate water to expand in the spring.
Bulk tissue water relations for leaves of R. ovata were not different from M. laurina and cannot explain the differential freezing tolerance of the two species. We hypothesized that greater capacitance might allow R. ovata to better tolerate periods of reduced water supply when Kl of stems was low compared to M. laurina. We also expected that R. ovata would have a lower osmotic potential at saturation (s(sat)) compared to M. laurina, which would depress the freezing point of its cells. However, we reject both of these hypotheses because capacitance and s(sat) were not different between the two species (Table 1).
The seedling stage of development often has the highest mortality and determines species persistence at a site; therefore, when attempting to understand the importance of an abiotic stress on the distribution of a plant species, it is often critical to demonstrate the effect of the stress on seedling establishment. Malosma laurina adults do not occur in the coldest valley bottoms at our cold study site, presumably because its seedlings are killed by frost. Numerous seedlings of R. ovata and M. laurina recruited in the cold valley bottoms at our cold site after the wildfire of 1996, providing us with a rare opportunity to assess seedling performance and survivorship during freeze–thaw stress. The results of this natural experiment were decisive, with seedlings of M. laurina showing frost damage following a –4.2°C freezing event and further damage and 100% mortality following a –7.2°C freezing event. In contrast to M. laurina seedlings at the cold site, co-occurring seedlings of R. ovata and control seedlings of M. laurina at a warm site showed no freezing damage and 100% of the R. ovata seedlings survived the subzero temperature events. These results clearly demonstrate the differential frost tolerance between seedlings of R. ovata and M. laurina and importantly show the role of freezing in limiting the distribution of M. laurina from colder microsites in southern California.
Freezing is often overlooked as a causal factor controlling species distributions in the Mediterranean climate region of southern California, where drought and wildfire receive more attention. In the context of climate change, freezing may be very important in southern California. Climate trends indicate that southern California has experienced 15% fewer nights with frost in the latter half of the 20th century (Christy et al., 2001
) and models predict that by 2030 to 2050 winters will be 3°C warmer than they are now (Field et al., 1999
). Malosma laurina populations in southern California often show strong recruitment from seed following wildfire or during moist conditions (Frazer and Davis, 1988
; Thomas and Davis, 1989
). As temperatures warm, M. laurina may expand its range into areas where it was previously limited by frost. Once past the second year of growth, M. laurina can resprout from a root crown following freezing-induced shoot death. The expansion of M. laurina into new microsites in the Santa Monica Mountains may be in progress.
In summary, the frost tolerance of R. ovata is largely explained by the ability of its leaves to tolerate freezing temperatures and desiccation and to minimize water loss from evergreen leaves when stems are highly embolized following freeze–thaw events. In contrast, M. laurina has none of these adaptations and suffers severe frost damage at the seedling and adult stages of development. Rhus ovata may be unique among evergreen woody plants in tolerating high levels of cavitation caused by freezing instead of avoiding cavitation, which based upon the literature, appears to be a more common strategy.
FOOTNOTES
1 The authors thank the National Science Foundation for supporting this research with grants IBN-0130870 and DBI-0243788. We thank Mike Takeshita and Bradley Yocum of the Malibu Forestry Unit, Forest Division of the Los Angeles County Fire Department, for supporting our field research. We thank two anonymous reviewers whose comments improved this manuscript. 
4 Author for correspondence (e-mail: bpratt{at}pepperdine.edu
)
LITERATURE CITED
Arora R. J. P. Palta 1988 In vivo perturbation of membrane-associated calcium by freeze–thaw stress in onion cells. Plant Physiology 87: 622-628
Ashworth E. N. R. S. Pearce 2002 Extracellular freezing in leaves of freezing-sensitive species. Planta 214: 798-805[CrossRef][Web of Science][Medline]
Boorse G. C. T. L. Bosma A.-C. Meyer F. W. Ewers S. D. Davis 1998a Comparative methods of estimating freezing temperatures and freezing injury in leaves of chaparral shrubs. International Journal of Plant Sciences 159: 513-521[CrossRef]
Boorse G. C. F. W. Ewers S. D. Davis 1998b Differential response of chaparral shrubs to below freezing temperatures. American Journal of Botany 85: 1224-1230
Campbell G. S. J. M. Norman 1998 An introduction to environmental biophysics. Springer-Verlag, New York, New York, USA
Carlquist S. W. D. A. Hoekman 1985 Ecological wood anatomy of the woody southern California flora. IAWA Bulletin 6: 319-347
Cavender-Bares J. N. M. Holbrook 2001 Hydraulic properties and freezing-induced cavitation in sympatric evergreen and deciduous oaks with contrasting habits. Plant, Cell and Environment 24: 1243-1256[CrossRef]
Christy J. R. R. A. Clarke G. V. Gruza J. Jouzel M. E. Mann J. Oerlemans M. J. Salinger S. W. Wang 2001 In J. T. Houghton, Y. Ding, D. J. Griggs, M. Noguer, P. J. van der Linden, X. Dai, K. Maskell, and C. A. Johnson [eds.], Climate change 2001: The scientific basis, 164–181. Cambridge University Press, New York, New York, USA
Cordero R. A. E. T. Nilsen 2002 Effects of summer drought and winter freezing on stem hydraulic conductivity of Rhododendron species from contrasting climates. Tree Physiology 22: 919-928[Abstract]
Davis S. D. F. W. Ewers J. S. Sperry K. A. Portwood M. C. Crocker G. C. Adams 2002 Shoot dieback during prolonged drought in Ceanothus (Rhamnaceae) chaparral of California: a possible case of hydraulic failure. American Journal of Botany 89: 820-828
Davis S. D. H. A. Mooney 1985 Comparative water relations of adjacent California shrub and grassland communities. Oecologia 66: 522-529[CrossRef][Web of Science]
Davis S. D. J. S. Sperry U. Hacke 1999 The relationship between xylem conduit diameter and cavitation caused by freeze–thaw events. American Journal of Botany 86: 1367-1372
DeSouza J. P. Silka S. D. Davis 1986 Comparative physiology of burned and unburned Rhus laurina after chaparral wildfire. Oecologia 71: 63-68[CrossRef][Web of Science]
Ewers F. W. M. C. Lawson T. J. Bowen S. D. Davis 2003 Freeze/ thaw stress of Ceanothus of southern California chaparral. Oecologia 136: 213-219[CrossRef][Web of Science][Medline]
Field C. B. G. C. Daily F. W. Davis S. Gaines P. A. Matson J. Melack N. L. Miller 1999 Confronting climate change in California: ecological impacts on the golden state, 1–63. Union of Concerned Scientists, Cambridge, Massachusetts, USA, and Ecological Society of America, Washington D.C., USA
Frazer J. M. S. D. Davis 1988 Differential survival of chaparral seedlings during the first summer drought after wildfire. Oecologia 76: 215-221[CrossRef][Web of Science]
Hickman J. C. 1993 The Jepson manual. University of California Press, Berkeley, California, USA
Kerstiens G. 1996 Cuticular water permeability and its physiological significance. Journal of Experimental Botany 47: 1818-1832
Koide R. T. R. H. Robichaux S. R. Morse C. M. Smith 1989 Plant water status, hydraulic resistance and capacitance. In R. W. Pearcy, J. Ehleringer, H. A. Mooney, and P. W. Rundel [eds.], Plant physiological ecology: field methods and instrumentation, 161–183. Champman and Hall, London, UK
Körner C. 1995 Leaf diffusive conductances in the major vegetation types of the globe. In E.-D. Schultze and M. M. Caldwell [eds.], Ecophysiology of photosynthesis, 463–490. Springer-Verlag, New York, New York, USA
Kubiske M. E. M. D. Abrams 1991 Rehydration effects on pressure– volume relationships in four temperate woody species: variability with site, time of season and drought conditions. Oecologia 85: 537-542[CrossRef][Web of Science]
Langan S. J. F. W. Ewers S. D. Davis 1997 Differential susceptibility to xylem embolism caused by freezing and water stress in two species of chaparral shrubs. Plant, Cell and Environment 20: 425-437[CrossRef]
Larcher W. 1995 Physiological plant ecology. Springer-Verlag, New York, New York, USA
Lipp C. C. E. T. Nilsen 1997 The impact of subcanopy light environment on the hydraulic vulnerability of Rhododendron maximum to freeze–thaw cycles and drought. Plant, Cell and Environment 20: 1264-1272[CrossRef]
Loik M. E. S. P. Redar 2003 Microclimate, freezing tolerance, and cold acclimation along an elevation gradient for seedlings of the Great Basin shrub, Artemesia tridentata. Journal of Arid Environments 54: 769-782[CrossRef][Web of Science]
Marinez-Vilalta J. W. T. Pockman 2002 The vulnerability to freezing-induced xylem cavitation of Larrea tridentata (Zygophyllaceae) in the Chihuahuan desert. American Journal of Botany 89: 1916-1924
Mayr S. F. Schwienbacher H. Bauer 2003 Winter at the alpine timberline. Why does embolism occur in Norway spruce but not in stone pine?. Plant Physiology 131: 780-792
Miller P. C. D. K. Poole 1979 Patterns of water use in shrubs in southern California. Forest Science 25: 84-98[Web of Science]
Percival G. C. A. Henderson 2003 An assessment of the freezing tolerance of urban trees using chlorophyll fluorescence. Journal of Horticultural Science and Biotechnology 78: 254-260
Pittermann J. J. Sperry 2003 Tracheid diameter is the key trait determining the extent of freezing-induced embolism in conifers. Tree Physiology 23: 907-914[Abstract]
Pockman W. T. J. S. Sperry 1997 Freezing-induced xylem cavitation and the northern limit of Larrea tridentata. Oecologia 109: 19-27[CrossRef][Web of Science]
Poole D. K. P. C. Miller 1981 The distribution of plant water stress and vegetation characteristics in southern California chaparral. The American Midland Naturalist 105: 32-43[CrossRef]
Rotman B. B. W. Papermaster 1966 Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proceedings of the National Academy of Sciences, USA 55: 134-141
Sakai A. W. Larcher 1987 Frost survival in plants: responses and adaptation to freezing stress. Springer-Verlag, New York, New York, USA
Sobrado M. A. 1997 Embolism vulnerability in drought-deciduous and evergreen species of a tropical dry forest. Acta Oecologia 18: 383-391[CrossRef]
Sparks J. P. R. A. Black 2001 Winter hydraulic conductivity and xylem cavitation in coniferous trees from upper and lower treeline. Arctic Antarctic Alpine Research 32: 397-403
Sperry J. S. J. R. Donnelly M. T. Tyree 1988 A method for measuring hydraulic conductivity and embolism in xylem. Plant, Cell and Environment 11: 35-40[CrossRef]
Sperry J. S. K. L. Nichols J. E. M. Sullivan S. E. Eastlack 1994 Xylem embolism in ring-porous, diffuse-porous, and coniferous trees of northern Utah and interior Alaska. Ecology 75: 1736-1752[CrossRef][Web of Science]
Sperry J. S. J. E. M. Sullivan 1992 Xylem embolism in response to freeze–thaw cycled and water stress in ring-porous, diffuse-porous, and conifer species. Plant Physiology 100: 605-613
Stiller V. J. S. Sperry 2001 Canny's compensating pressure theory fails a test. American Journal of Botany 86: 1082-1086[CrossRef]
Stratton L. G. Goldstein F. C. Meinzer 2000 Stem water storage capacity and efficiency of water transport: their functional significance in a Hawaiian dry forest. Plant, Cell and Environment 23: 99-106[CrossRef]
Thomas C. M. S. D. Davis 1989 Recovery patterns of three chaparral shrub species after wildfire. Oecologia 80: 309-320[CrossRef][Web of Science]
Tyree M. T. F. W. Ewers 1991 The hydraulic architecture of trees and other woody plants. New Phytologist 119: 345-360[CrossRef][Web of Science]
Tyree M. T. H. T. Hammel 1972 The measurement of the turgor pressure and water relations of plants by the pressure bomb technique. Journal of Experimental Botany 23: 267-282
Vankat J. L. 1989 Water stress in chaparral shrubs in summer-rain versus summer-drought climates—-whither the Mediterranean-type climate paradigm?. In S. Keeley [ed.], The California chaparral: paradigms reexamined, 117–124. Natural History Museum of Los Angeles County, Los Angeles, California, USA
Windholm J. M. 1972 The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technology 47: 189-194[Web of Science][Medline]
Yang S. M. T. Tyree 1992 A theoretical model of hydraulic conductivity recovery from embolism with comparison to experimental data on Acer saccharum. Plant, Cell and Environment 15: 633-643[CrossRef]
CiteULike Complore Connotea Del.icio.us Digg Facebook Reddit Technorati Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  F. Valladares, J. Zaragoza-Castells, D. Sanchez-Gomez, S. Matesanz, B. Alonso, A. Portsmuth, A. Delgado, and O. K. Atkin Is Shade Beneficial for Mediterranean Shrubs Experiencing Periods of Extreme Drought and Late-winter Frosts? Ann. Bot., December 1, 2008; 102(6): 923 - 933. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Jacobsen, F. W. Ewers, R. B. Pratt, W. A. Paddock III, and S. D. Davis Do Xylem Fibers Affect Vessel Cavitation Resistance? Plant Physiology, September 1, 2005; 139(1): 546 - 556. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), the leaf water potential at the turgor loss point (
