Research
Foliar uptake of fog water and transport belowground alleviates
drought effects in the cloud forest tree species, Drimys
brasiliensis (Winteraceae)
Cleiton B. Eller, Aline L. Lima and Rafael S. Oliveira
Department of Plant Biology, Institute of Biology, University of Campinas – UNICAMP, CP6109, Campinas, S~ao Paulo, Brazil
Summary
Author for correspondence:
Rafael S. Oliveira
Tel: +55 1935216177
Email: [email protected]
Received: 19 December 2012
Accepted: 24 February 2013
New Phytologist (2013)
doi: 10.1111/nph.12248
Key words: drought, fog, hydraulic failure,
hydraulic redistribution, sap flow, soil–plant–
atmosphere continuum, stable isotopes,
tropical cloud forests.
Foliar water uptake (FWU) is a common water acquisition mechanism for plants inhabiting
temperate fog-affected ecosystems, but the prevalence and consequences of this process for
the water and carbon balance of tropical cloud forest species are unknown.
We performed a series of experiments under field and glasshouse conditions using a combination of methods (sap flow, fluorescent apoplastic tracers and stable isotopes) to trace fog
water movement from foliage to belowground components of Drimys brasiliensis. In addition, we measured leaf water potential, leaf gas exchange, leaf water repellency and growth
of plants under contrasting soil water availabilities and fog exposure in glasshouse experiments to evaluate FWU effects on the water and carbon balance of D. brasiliensis saplings.
Fog water diffused directly through leaf cuticles and contributed up to 42% of total foliar
water content. FWU caused reversals in sap flow in stems and roots of up to 26% of daily
maximum transpiration. Fog water transported through the xylem reached belowground
pools and enhanced leaf water potential, photosynthesis, stomatal conductance and growth
relative to plants sheltered from fog.
Foliar uptake of fog water is an important water acquisition mechanism that can mitigate
the deleterious effects of soil water deficits for D. brasiliensis.
Introduction
The occurrence of frequent fog events is a defining climatic attribute
of tropical montane cloud forests (TMCFs), with multiple and yet
poorly understood ecological effects (Bruijnzeel & Veneklaas,
1998). The direct contact of fog water droplets with the surface
of stems and leaves causes water to drip to the soil, and this additional precipitation is considered to be a major hydrological input
in TMCFs (Bruijnzeel et al., 2011; Cavelier et al., 1996; Holder,
2006). In addition, a substantial proportion of the fog intercepted by tree crowns may be retained by the foliage and subsequently evaporate back to the atmosphere, or potentially
be absorbed by leaves. Foliar water uptake (FWU) of fog has been
widely reported in several temperate ecosystems (Burgess &
Dawson, 2004; Breshears et al., 2008; Limm et al., 2009;
Simonin et al., 2009), and, with the exception of a recent study
in Costa Rican TMCFs (Goldsmith et al., 2013), the prevalence
and ecophysiological consequences of this water acquisition
mechanism in TMCF tree species remain largely unexplored.
Leaf wetting events can increase the water status of plants when
water is absorbed by leaves (Grammatikopoulos & Manetas,
1994; Yates & Hutley, 1995; Breshears et al., 2008), but may be
detrimental to photosynthesis as the presence of a water film over
a leaf reduces the diffusion velocity of CO2 by c. 104 times
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(Smith & McClean, 1989; Brewer & Smith, 1997). As a consequence, a strong selective pressure for high leaf surface hydrophobicity should be expected in plants inhabiting very wet
environments (Smith & McClean, 1989; Brewer & Smith, 1997;
Feild et al., 1998). However, empirical data have shown that leaf
water repellency is, in fact, lower in environments often subject
to leaf wetting events (Holder, 2007; Rosado et al., 2010). Furthermore, a recent study in redwood forests has demonstrated
that fog interception and FWU can decouple leaf-level gas
exchange from the soil and have a positive effect on carbon balance when plants are under soil water deficits (Simonin et al.,
2009).
TMCFs occur under a wide range of annual rainfall regimes
(from 600 to 4500 mm), and some forests occur under climates
with a marked seasonal drought ( Jarvis & Mulligan, 2011).
Moreover, during periods when fog is absent, high-altitude
environments in tropical regions are considered to be arid environments for plants because of their high potential evapotranspiration caused by the high radiation load and atmospheric
demand (Leuschner, 2000). On top of this, climate models suggest that global warming is causing an upward shift of the cloud
basis for most of the world’s TMCFs, which will impose drier
conditions for TMCF species in the near future (Still et al.,
1999). Therefore, experimental research on how fog affects plant
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performance is crucial for our understanding of how TMCF tree
species will respond to future drier climates.
In the present study, we assessed the ecological importance of
FWU for Drimys brasiliensis Miers. (Winteraceae), an abundant
and widely distributed woody species in the Atlantic cloud forests
of Brazil. We hypothesized that the foliar uptake of fog water is an
important water acquisition mechanism for D. brasiliensis which
can improve plant water status and favor the plant’s carbon balance. We conducted a suite of experiments using a combination
of sap flow, stable isotopes, apoplastic tracers and ecophysiological
measurements under field and glasshouse conditions to investigate how fog and FWU affect the water and carbon balance of
D. brasiliensis. In addition, our study provides information on
the anatomical pathways involved in the process of FWU and
novel insights into plant water dynamics. We demonstrate, for
the first time, that fog water is not only absorbed by leaves, but
also transported through the xylem to plant belowground
components (plant roots and, probably, mycorrhizal hyphae and
rhizosphere soil) when plants are simultaneously exposed to fog
events and dry soil.
Materials and Methods
Field observations
Study area Field observations were carried out in a cloud forest
stand, at Campos do Jord~ao State Park (CJSP, 22°69′S, 45°52′W),
located in the Mantiqueira Range, 15 km east of the town of
Campos do Jord~ao, S~ao Paulo, Brazil. The forest stand is located
at an altitude of c. 2000 m, characterized by a low canopy height
(maximum of 12 m) and a contrasting floristic assemblage compared with lowland evergreen forests (Bertoncello et al., 2011).
Drimys brasiliensis Miers. is an overstory and evergreen tree that
is extensively distributed in Atlantic cloud forests (Safford, 1999;
Bertoncello et al., 2011) and is the most abundant tree species in
our site.
Mean annual rainfall at the site is 1705 mm with a distinct dry
period of 3 months ( June–August), in which < 8% of total
annual precipitation falls, indicating pronounced seasonality of
rainfall (Center for Meteorological and Climate Research
Applied to Agriculture, 2013). Dry spells are frequent in the
Mantiqueira region and, in some years, can be very long and
severe, especially in years under El Ni~
no-Southern Oscillation
influence (Safford, 1999). The mean annual temperature is
14.9°C and July is the coldest month (mean of 10.3°C), with
minimum temperatures frequently falling to zero (CEPAGRI,
2012). Fog occurs on 65–90% of the days in cloud forests of the
Mantiqueira range (Segadas-Vianna & Dau, 1965), as they are
under the influence of subtropical, temperate and polar air masses
(Safford, 1999). These polar masses are more frequent and
intense during the dry season (June–September), and are responsible for intense fog and weak rain events (Safford, 1999).
Sap flow To investigate how fog affects the direction of water
flow in the vascular system of D. brasiliensis, we monitored sap
flow in stems and roots of five adult trees (c. 8–12 m height) in
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the cloud forest stand using the heat ratio method (HRM; Burgess et al., 2001). Reversals in the direction of sap flow during fog
events were used as evidence of FWU. The sensors (ICT International, Armidale, NSW, Australia) were installed at a height of c.
60–90 cm in the stems and at c. 10–40 cm below the soil surface
in the roots. The sensors emitted heat pulses every 30 min and
the measurements were stored in a data logger (model SL5 Smart
logger; ICT International). We calculated the vapor pressure deficit (VPD) at the site using data collected by air humidity and
temperature sensors (model U23-001; Onset Computer Corporation, Bourne, MA, USA). Leaf wetness and precipitation data
were also measured using leaf wetness sensors and pluviometers
(models S-LWA-M003 and RG3-M, respectively; Onset Computer Corp.). All these sensors were connected to a data logger
(model H21-002; Onset Computer Corp.). We used these
micrometeorological data to infer fog events: when VPD was
close to zero, leaves were wet and no rain was being collected in
the pluviometers, this was considered a period of fog. At the end
of the study, we interrupted sap flow in two stems by drilling
holes to form a 3-cm-wide cut immediately above and below the
probes. We used the values collected by the sensors after the flow
was interrupted as our reference zero, so that we could correct the
data using the procedures described in Burgess et al. (2001).
Based on these data, we established the micrometeorological conditions when zero flow occurred, which allowed us to determine
the baseline for the sensors that did not have their flow interrupted. More details on this approach can be found in Burgess &
Dawson (2004) and Rosado et al. (2012).
Branch spraying experiment To evaluate the short-term effects
of FWU on leaf water relations, we conducted an experiment by
spraying water onto cut branches at CJSP, based on the protocol
used by Breshears et al. (2008). Two branches under similar
micrometeorological conditions were collected from 12 individuals. One branch of each pair was sprayed with water (SP: spray
treatment) and the other was kept dry (CT: control); both were
kept in separate dark plastic bags for 1–2 h. Foliar water content
(FWC) was determined for 10 mature leaves after the treatments,
and leaf water potential (ΨL) was measured on two to three samples before and after the treatment. We calculated the final FWC
(%) as: ((weight of fresh leaf weight of dry leaf)/weight of fresh
leaf) 9 100. We used a Scholander pressure bomb (Model 1000;
PMS, Corvallis, OR, USA) to measure ΨL.
Laboratory experiment
Apoplastic tracer experiment To evaluate the anatomical pathways involved in foliar uptake, we performed a laboratory experiment exposing leaves of D. brasiliensis to a fluorescent apoplastic
tracer solution. For this experiment, we used fresh, mature and
detached leaves from D. brasiliensis saplings collected at CJSP
and kept in a glasshouse. The cut portion of the leaves was
sealed with parafilm and maintained in a dark moist chamber
(Mastroberti & Mariath, 2008), where it remained in contact
with 100 ll of 1% Lucifer Yellow carbohydrazide dilithium salt
aqueous solution (LY; Sigma-Aldrich) for 24 h. This tracer is
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nontoxic to plant tissues, and can only move through apoplastic
pathways (Oparka & Read, 1994). Leaves were then washed in
distilled water, carefully dried with filter paper, hand sectioned
and prepared for microscopic observation in a 90% glycerolphosphate buffer (Mastroberti & Mariath, 2008). Sections were
observed using epifluorescence (Leica DFC500M-R; Wetzlar,
Germany), under intense blue excitation of 450–490 nm with a
515-nm barrier filter (Oparka & Read, 1994). Classical anatomical assays were also conducted to determine the chemical composition of leaf structures and possible hydrophilic pathways in the
leaves. Mature leaves collected at CJSP and fixed in 50% ethyl
alcohol–formaldehyde–acetic acid (FAA; Johansen, 1940) were
embedded in plastic resin (Historesin®; Leica Biosystems,
Wetzlar, Germany), and then sectioned. These sections were then
stained with Sudan Black, to identify lipophilic cuticular structures (Pearse, 1980), Periodic Acid-Schiff reaction (PAS) to identify hydrophilic polysaccharide compounds, such as mucilages,
glycogen, glycolipids and glycoproteins (McManus, 1948), and
Red Ruthenium to identify pectins (Johansen, 1940).
Glasshouse observations
We performed a series of four experiments in a glasshouse at the
University of Campinas (Campinas, Brazil) from June 2009 to June
2011 to evaluate how fog affects the water and carbon relations,
growth, survival and leaf water repellency of D. brasiliensis, and also
to trace fog water movement from foliage to belowground components of plants. Plants in the glasshouse were subjected to natural
daily and seasonal cycles of solar radiation (with a daily peak of
photosynthetically active radiation (PAR) of 470–840 lmol
photons m 2 s 1), natural temperature (8–39°C) and relative
humidity (10–100%).
E1. Ecophysiological performance experiment To assess the
effects of regular fog exposure on the ecophysiological performance of D. brasiliensis, we conducted a glasshouse experiment
with saplings of D. brasiliensis (20–60 cm in height) collected at
CJSP and planted in 34-l pots. One month after being transplanted, plants were randomly assigned to control (well watered),
fog and drought treatments. Before the start of the experiment,
all plants were watered until the soil reached field capacity. The
total number of plants was 24 for the control, 25 for the fog and
24 for the drought treatments. Within this pool of plants, two
subsets were assigned for destructive and nondestructive measurements, as described in the sections below. In the fog treatment,
we exposed the shoots of all plants to 8 h of artificial fog inside
fog chambers made of PVC and plastic. We submitted the plants
to nocturnal artificial fog three times per week for 2 months during the spring of 2010 (September–October). Fog was generated
by an ultrasonic device (model Waterclear Premium; Soniclear,
S~ao Paulo, Brazil), which produces a water aerosol with droplets
smaller than 20 lm. The soil in pots was completely sealed with
plastic bags and parafilm to prevent fog water from reaching the
soil. Watered plants received water on the soil until saturation
every day, whereas drought-affected plants were not irrigated during the experiment. We measured the ecophysiological
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parameters seven times during the 2 months of the experiment,
and also monitored environmental variables (VPD and relative
water content (RWC)) inside the fog chambers. VPD was calculated using the data collected by air humidity and temperature
sensors (model U23-001; Onset Computer Corp.); RWC was
measured gravimetrically on three soil samples randomly chosen
within each treatment. To avoid excessive removal of soil in some
pots, different experimental units were used during the experiment. Soil samples were collected weekly using a PVC cylinder
(10 mm) from the surface to the bottom of the pot, so that every
sample contained a full depth profile of the soil. All samples were
weighed immediately after collection and RWC was calculated
as: ((weight of fresh soil weight of dry soil)/weight of fresh
soil) 9 100.
We measured leaf water potential at predawn (ΨPD) and at
midday (ΨMD) for three to eight plants per treatment, randomly
selected from a group of 13 individuals per treatment. This group
of 13 plants per treatment was used only for destructive measurements with the Scholander pressure bomb. Several saplings had
only three to five leaves, and so we used this sampling method for
water potential measurements to minimize drastic reductions in
the leaf area of the saplings.
We measured the instantaneous maximum net CO2 assimilation rate (Amax) and stomatal conductance (gs) using an infrared
gas analyzer (ADC BioScientific LCpro+; Analytical Development Company, Hoddesdon, Hertfordshire, UK) in mature,
fully expanded leaves from a group of five plants for the fog
treatment and four plants for the drought and control treatments. Measurements were taken between 08:00 and 09:30 h,
the period of greatest photosynthetic activity, as determined by
a daily photosynthesis curve for this species performed just
before the beginning of the experiment. Leaf chamber PAR was
controlled at 900 lmol photons m 2 s 1, which was sufficient
to light saturate the species A, according to a light response
curve that was obtained before the experiment. VPD, CO2 and
the temperature of the IRGA cuvette were kept at ambient
levels.
We measured the stem diameter (hst), height (h) and estimated total leaf area at the beginning and end of the experiment on five control plants and six drought and fog plants.
None of these plants was submitted to destructive measurements. The stem diameter was measured with a digital caliper
(Mitutoyo Sul Americana Ltda, Suzano, Brazil) at the base of
the stem, and plant height was measured from the base of
the stem to the highest leaf with a metric tape. To calculate
the total leaf area, we measured the area of 25 leaves with
ImageJ 1.42 and used a linear regression between the measured leaf area (y) and the product between each leaf length
and width (x). We used the equation derived from the linear
model (y = 0.909x + 0.7504; R2 = 0.98) to predict the area of
the other leaves and obtained the total leaf area by summing
the areas of every leaf in the plant.
To obtain a proportional rate of growth or loss in total leaf
area, we divided the final values by the initial values of these variables. Mortality rates of the plants that were not submitted to
destructive measurements (12 plants in the fog treatment and 11
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plants in the control and drought treatments) were determined at
the end of the experiment.
E2. Sap flow and leaf hydrophobicity experiment To evaluate
how fog affects the direction of water flow in the vascular system
of D. brasiliensis, we monitored sap flow in stems of 12 saplings
(70–140 cm in height) collected at CJSP and planted in 88-l
pots, in a second glasshouse experiment. We installed HRM sensors at the base of the stems, and exposed four of each of these
plants to the same treatments as described in the previous experiment (control, drought and fog). We calculated the reference zero
flow value for these plants using the VPD data collected with air
humidity and temperature sensors (model U23-001; Onset
Computer Corp.) kept inside the fog chamber. We assumed that
the sap flow was zero during nights with low VPD and no leaf
wetness (Rosado et al., 2012). Sap flow velocity was calculated
according to Burgess et al. (2001). Sap flow data are expressed as
the percentage of the maximum value reached by each sensor
during the experiment.
In addition, we measured leaf hydrophobicity for three leaves
from each plant, before and after the treatments, to assess how
fog affects leaf surface wettability. We compared the changes in
leaf hydrophobicity of the four plants exposed to fog with the
group of plants not exposed to fog (control treatment, N = 4).
We measured the contact angle of a 5-ll droplet of distilled
water on both sides of the leaf surfaces. We fixed leaves on a horizontal flat surface and photographed the droplet resting on each
side of the leaf surface. We used the software ImageJ 1.44 to analyze the contact angle of the leaf and the water droplet, using the
same principle as described by Aryal & Neuner (2010).
E3. Deuterium labeling experiments To evaluate the amount
of water absorbed by FWU, we exposed the shoots of the saplings
to deuterium-enriched fog during the night in a third experiment
in the fog chamber. Soil irrigation was suppressed 1 wk before
the beginning of the fog treatment. The isotopic composition
was expressed in delta notation (dD&) relative to the V-SMOW
standard. Labeled water (c. 668& dD) used in the fog treatment
was composed of a mixture of tap water (c. 44& dD) and water
enriched in deuterium oxides (99.8%; from Cambridge Isotope
Laboratory, Andover, MA, USA). The isotopic enrichment of
fogged leaves compared with control leaves was used as evidence
of foliar uptake, as soils and roots were completely isolated from
the fog by plastic bags and parafilm. We used the IsoError mixing
model to estimate the contribution of fog to leaf water content
(Phillips & Gregg, 2001).
Before the beginning of each fog treatment, leaves from control and treatment groups were carefully collected at 17:30 h,
washed with tap water, dried with paper towels and kept sealed
in vials with parafilm at 20°C. The same procedure was performed the following morning at 07:30 h, after the plants had
been exposed to nocturnal fog. Leaf wetness was monitored using
a leaf wetness sensor (S-LWA-M003; Onset Computer Corp.) to
ensure that the fog chamber reached saturation as soon as nebulization started, to avoid the potential occurrence of water diffusion back to the atmosphere via stomata (Limm et al., 2009).
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Because of the magnitude of FWU and sap flow reversals
observed in D. brasiliensis and the effect of the fog treatment on
soil RWC (in the above-mentioned experiments), we hypothesized that D. brasiliensis might transport a significant amount of
water absorbed by the shoots to belowground pools. To test
whether fog water was actually being transported belowground,
we performed a fourth experiment, exposing six D. brasiliensis
saplings to fog water enriched in deuterium (c. 295& dD). We
then compared the isotopic enrichment of soil water samples
(including fine roots) collected from rhizosphere soil in sealed
pots after three sequential nocturnal nebulizations, between
plants exposed to enriched fog and plants exposed to nonenriched fog (control). We opted to use plants exposed to nonenriched fog as the control in order to impose similar nocturnal
micrometeorological conditions for both groups and to minimize
the occurrence of night-time transpiration or soil evaporation
events that could cause evaporative enrichment of the stable isotope composition of soil water. Tap water was used to generate
fog for control plants and to irrigate plants before the experiment.
Because soil and roots were isolated from the fog, we considered
the isotopic enrichment of soil water (dD) after the nebulizations
as evidence that water was being transported belowground. Soil
samples from the pot of each plant were collected from three different spots in the pot using a metal pipe of 10 mm in diameter.
The samples were collected from the surface to the bottom of the
pot (c. 35 cm), so that every sample contained a full depth profile
of the soil. The most superficial layer of soil (c. 2–3 cm) was discarded to ensure that the sample was not contaminated by any
dripping or subjected to evaporative enrichment. The samples
were kept sealed in vials with parafilm and frozen before analysis.
Water from leaf and soil samples was extracted by cryogenic distillation at Laboratorio de Ecologia Isotopica (CENA/USP,
Piracicaba, Brazil). Subsequently, these water samples were
analyzed in a DeltaPlus Advantage mass spectrometer (intrinsic
error c. 0.5& ; Thermo Finningan, San Jose, CA, USA) at
CPGeo/USP, S~ao Paulo, Brazil.
Statistical analysis
Responses were analyzed for the ecophysiological experiment
using a repeated-measures mixed-model ANOVA using PROC
MIXED (SAS v9.3; SAS Institute, Cary, NC, USA), with treatment and time as fixed factors. We used a one-way ANOVA to
compare the effects of treatments on growth data using Systat 11
(Systat Software Inc., Richmond, CA, USA). Data were tested
for normality and homogeneity of variance and, when necessary,
log (base 10) transformed before the analysis. Bonferroni post-hoc
tests were conducted when the effects were significant (P < 0.05),
to assess the differences between treatments.
Responses for the isotope, branch spraying and leaf wettability
experiments were analyzed using t-tests, adjusted for dependent
samples and unequal variances when necessary. We used a pairwise Fisher’s exact test (with Bonferroni correction for multiple
comparisons) to assess the effects of treatments on plant mortality. These tests were performed using R v.2.15.1 (R Development
Core Team, 2012).
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Results
Foliar water uptake
We demonstrate by several lines of evidence that foliar uptake of
fog water is an important water acquisition mechanism for
D. brasiliensis. First, we observed that the LY solution diffused
directly through leaf cuticles of both surfaces (Fig. 1a–d), moved
through apoplastic routes and was retained by parenchymatic
cells (Fig. 1c,d). We found that leaves of D. brasiliensis were rich
in hydrophilic compounds (Fig. 1f–h), which may have contributed to water absorption and the retention of apoplastic tracers in
the mesophyll of this species. Leaf surfaces were rich in polysaccharides (Fig. 1f ), and cell walls of palisade parenchyma closer to
the midrib and leaf edges were rich in acidic pectins (Fig. 1g,h).
These hydrophilic compounds probably influenced the low
leaf surface hydrophobicity observed. Leaves of D. brasiliensis had
a mean contact angle of c. 44° (adaxial surface) and c. 71° (abaxial
surface), being considered highly wettable (contact angles < 90°),
according to the classification used by Aryal & Neuner (2010).
The leaf adaxial surface was significantly more wettable than the
abaxial surface (paired t-test: t = 6.879, df = 15, P < 0.001;
Fig. 2). Leaves of plants exposed to regular fog events showed a
substantial decrease in their abaxial surface hydrophobicity,
whereas leaves not exposed to fog became less wettable (t-test:
t = 4.230, df = 6, P = 0.005; Fig. 2a). Fog had no effect on the
adaxial leaf surface hydrophobicity (t-test: t = 0.285, df = 6,
P = 0.785; Fig. 2b).
Results from our branch scale experiments in the field and in
the glasshouse provided further evidence that water was directly
absorbed by D. brasiliensis leaves. Significant amounts of deuterium (D) were traceable in leaves exposed to deuterium-enriched
fog: c. 42% of fog water D was found in the leaves within 8 h
(Fig. 3a). FWU increased the FWC of sprayed D. brasiliensis
leaves by 2.1% (absolute value) in comparison with control nonsprayed branches (t = 18.454, P < 0.001), and this resulted in an
increase in ΨL of 0.39 MPa (Fig. 3b).
Fog internal hydraulic redistribution
We observed reversals of sap flow in stems and roots in every
adult plant monitored in the field during fog events (Fig. 4a,b);
reversals of sap flow caused by fog happened not only during the
night-time, but also during the day (Fig. 4a,b). Similar patterns
of sap flow reversals were observed in plants exposed to fog in the
glasshouse experiments (Fig. 4c,d). In this experiment, paired
plants that were not exposed to fog did not show any sap flow
reversals. The magnitude of sap flow reversals was higher in
plants in the glasshouse experiment (Fig. 4c), reaching up to 26%
of the diurnal sap velocity, compared with 10% in adult plants in
(a)
(d)
(c)
(b)
(e)
(f)
(g)
(h)
Fig. 1 Transverse sections of leaves exposed to a solution of Lucifer Yellow carbohydrazide (CH) dilithium salt to 1% (LY) test for 24 h and hydrophilic
compounds in the leaf surfaces of Drimys brasiliensis. (a) Autofluorescence of fresh adaxial leaf surface (untreated leaves). (b) Autofluorescence of fresh
abaxial leaf surface (untreated leaves) (c) LY apoplastic fluorescent tracer concentrate in cell walls of the palisade parenchyma. (d) LY abaxial epidermis and
spongy parenchyma with apoplastic tracer. (a–d) Cut freehand under Intense Blue Filter between 450 and 490 nm; barrier, 515 nm. (e) Cuticle on both leaf
surfaces, with intrusion through some stomatal ostioles on adaxial surface (cuticular flange; Black Sudan reaction). (f) Epidermis and parenchyma from both
surfaces full of polysaccharides (Periodic Acid-Schiff reaction in dark pink; unstained leaf section detail on the left). (g) Cell walls below central vessel from
midrib rich in pectin compounds, as in (h) cells from leaf edge (g, h, pectin compounds have a light pink coloration given by the Red Ruthenium stain).
Arrow, stomatal aperture; Ct, cuticle; Ep, uniseriate epidermis; Id, idioblast; PP, palisade parenchyma; SP, spongy parenchyma. Bars: (a–d) 5 mm; (e–h)
25 lm.
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(a)
(b)
Fig. 2 Changes in leaf wettability on the abaxial surface (a) and adaxial surface (b) in Drimys brasiliensis saplings not exposed to fog (Control; n = 4) and
saplings regularly exposed to fog for 3 months (Fog treatment; n = 4). Leaf abaxial surface hydrophobicity was decreased significantly in plants exposed to
fog, whereas leaves not exposed to fog became less wettable (t = 4.230, df = 6, P < 0.01). Fog had no effect on the adaxial leaf surface hydrophobicity
(t = 0.285, df = 6, P = 0.785). The horizontal lines represent the median, and the top and bottom of the box the 25th and 75th quartiles. Whiskers
represent the 1.5 interquartile range.
Fig. 3 Shoot-scale experiments assessing foliar absorption in Drimys brasiliensis using control shoots and shoots exposed to fog. (a) Deuterium enrichment
of leaf water (dD, &) in plants exposed to nocturnal deuterium-enriched fog (n = 6) and in control plants (n = 6). Note the higher deuterium enrichment in
the leaf water of the plants exposed to deuterium-enriched fog (t-test: t = 5.8062, df = 5.071, P = 0.002). (b) Changes in ΨL (MPa) of cut branches after
being sprayed with water. The sprayed branches (n = 13) had a higher ΨL than the control branches (n = 13; t-test: t = 7.6432, df = 21.659, P < 0.001).
The horizontal lines represents the median, and the top and bottom of the box the 25th and 75th quartiles. Circles represent values outside the 1.5
interquartile range (whiskers).
the field. Most of the negative sap velocity values observed during
the fog events were higher than the resolution limit of the HRM
sensors (0.5 cm h 1).
We confirmed that fog water reached plant belowground components (roots and probably the rhizosphere) with our isotopic
tracing experiment. dD of soil water collected close to
D. brasiliensis roots increased more significantly in plants exposed
to deuterium-enriched fog than in control plants (Fig. 5). Fog
water contributed an average of 6.8 3.1% (mean SE) to the
rhizosphere soil water.
Ecophysiological performance experiment
At the beginning of the experiment, there was no difference in
ΨPD between the treatment groups and the means of all treatments ranged from 0.16 to 0.21 MPa (Fig. 6a). After 45 d,
plants regularly exposed to fog maintained ΨPD similar to control
levels, with mean values of 0.12 and 0.17 MPa in the water
and fog treatments, respectively. At this time, plants under the
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drought treatment had a ΨPD significantly more negative than
that of the control group (P = 0.012), with a mean value of
0.6 MPa. At the end of the experiment (after 59 d), fogged
plants still maintained similar ΨPD to control plants. The treatments did not affect ΨMD significantly (Table 1, Fig. 6b).
Gas exchange parameters (Amax and gs) were affected by the
treatments and by the interaction between treatments and time
(gs was only marginally affected by the interaction between treatment and time at P = 0.07; Table 1, Fig. 6c,d). All the treatments
started with similar Amax (Fig. 6c,d), with mean values ranging
from 4.5 to 5.7 lmol CO2 m 2 s 1, and, at the end of the experiment, plants in the drought treatment had a lower Amax (mean of
0.22 lmol CO2 m 2 s 1) compared with the other treatments
(P < 0.05). Amax values for the fog and control groups were not
significantly different at the end of the experiment (means of 2.9
and 5.2 lmol CO2 m 2 s 1, respectively). The gs value of plants
subjected to the fog treatment was not significantly different from
the control (means of 0.07 and 0.12 mol H2O m 2 s 1 in the fog
and control treatments, respectively). Towards the end of the
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(a)
(c)
(b)
(d)
Fig. 4 Sap flow patterns during fog events in saplings and adult individuals of Drimys brasiliensis. Data were normalized to allow comparisons among
individuals of different size and parts of trees with different flow magnitudes. (a) Stem and root sap flow in adult D. brasiliensis during the dry season (21–24
July). Sap flow values are the means of four stems and three roots of different D. brasiliensis plants under field conditions. Black bars indicate fog events,
inferred from micrometeorological data. (b) Micrometeorological data on field conditions during sap flow data collection. Conditions of high air humidity
(vapor pressure deficit (VPD) close to zero), wet leaves (leaf wetness (LW) reaching 100%) and no rain (data not shown) were assumed to be fog events. (c)
Stem sap flow of two D. brasiliensis saplings during the glasshouse experiment. Black bars indicate when plants were submitted to artificial fog. Note the
high-magnitude flow reversals during fog events. (d) Micrometeorological conditions in the glasshouse during sap flow data collection. During nebulizations,
the VPD value was zero and LW was 100%, suggesting that the artificial nebulizations created conditions similar to those observed in the field.
Fig. 5 Changes in soil water dD in Drimys brasiliensis exposed to
deuterium-enriched fog (Fog treatment; n = 6) and in plants exposed to
nonenriched fog (Control; n = 4) in a glasshouse experiment. Data show
significant deuterium enrichment of soil water in plants exposed to
deuterium-enriched fog after three sequential nocturnal nebulizations
(t-test: t = 2.36, df = 8, P = 0.04). Note that the soil water dD data
presented here are probably a mixture of water from soil, fine roots and
mycorrhizal hyphae. The horizontal lines represents the median, and the
top and bottom of the box the 25th and 75th quartiles. Whiskers represent
the 1.5 interquartile range.
experiment, gs values of plants exposed to fog remained similar to
those of the control group, with means of 0.04 and 0.07 mol
H2O m 2 s 1, respectively. The drought treatment was the only
treatment that showed significantly lower gs values at the end of
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the experiment (P = 0.019), beginning the experiment with a
mean of 0.10 mol H2O m 2 s 1 and decreasing to 0.01 mol
H2 O m 2 s 1 .
Soil RWC was also affected by the water treatments and by the
interactions between treatments and time (Table 1, Fig. 6e). At
the beginning of the experiment, RWC was not significantly
different among the treatments (P > 0.1), and, at the end of the
experiment, only RWC of fogged and control treatments
remained not significantly different. In the drought treatment,
RWC decreased after the start of the imposed drought, and
remained lower for that treatment (P < 0.001), being 14.6% at
the end of the experiment.
Finally, fog positively affected the growth and survival of
D. brasiliensis (Fig. 7). Fogged plants had similar stem and height
growth to control plants, whereas plants from the drought treatment showed less growth than plants in the other treatments
(P < 0.05; Fig. 7a). Plants in the drought treatment had completely lost their leaves by the end of the experiment, whereas
fogged plants decreased their leaf area (but maintained some leaf
area) and control plants increased their leaf area (Fig. 7c). The
survival of D. brasiliensis plants under fog and control treatments
was not significantly different, with mortalities of 9.1% and 25%
in the control and fog treatments, respectively. Plants under
drought showed a lower survival rate than that of the control
treatment (P = 0.022; Fig. 7d), with a mortality of 74% by the
end of the experiment.
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8 Research
(a)
Table 1 Mixed-model repeated measures analyses of the effects of
treatment through time on Drimys brasiliensis ecophysiological and environmental response variables
Source
Treatment
(b)
Treatment
9 time
Response
variable
F value
Numerator
df
Denominator
df
P value
ΨPD
ΨMD
A
gs
RWC
ΨPD
ΨMD
A
gs
RWC
3.31
0.61
9.82
4.10
57.24
1.56
0.66
2.73
1.72
3.30
2
2
2
2
2
14
14
14
14
14
30
31
11
11
44
64
63
71
71
41
0.051
0.549
0.003
0.046
< 0.001
0.12
0.782
0.002
0.071
0.001
P values in bold are significant at a = 0.05.
(c)
Discussion
Foliar water uptake
(d)
(e)
Fig. 6 Temporal dynamics of ecophysiological parameters of Drimys
brasiliensis and relative soil water content during the glasshouse
experiment. Points represent the means SE. Dashed line - Control; gray
line - Drought, solid line - Fog. (a, b). Predawn and midday leaf water
potential (ΨPD and ΨMD; measurements of leaf water potential on the 51st
and 59th days could not be performed for the drought treatment, because
the petioles collapsed at pressures above 2.5 MPa). (c) Rate of net CO2
assimilation (A), (d) stomatal conductance (gs), (e) relative water content
in the soil (RWC).
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In this study, we have demonstrated, by several lines of evidence,
that foliar uptake of fog water is an important water uptake
mechanism that positively affects the water and carbon balance of
D. brasiliensis. In addition, our study provides information on
the anatomical pathways involved in the process of FWU. Our
apoplastic tracer experiment suggested that large portions of the
abaxial and adaxial surfaces of leaves were involved in water
absorption, instead of a single specialized leaf structure (Fig. 1a–d).
Drimys brasiliensis has a mesophyll rich in hydrophilic phenolic compounds, polysaccharides and mucilage cells that have
already been suggested to be involved in the foliar uptake and
retention of water in leaves of Araucaria angustifolia (Mastroberti
& Mariath, 2008). We also observed a high concentration of
polysaccharides in the epidermis of both leaf surfaces and the
presence of pectins in the palisade parenchyma cell walls – structures that retained the fluorescent salt solution (Fig. 1c,d).
The abaxial surfaces of leaves have a papillose epidermis covered by a thick cuticle and lipophilic stomatal plugs that fill the
cavity above the guard cells (Feild et al., 1998; Fig. 1e). The function of these structures was first thought to be related to adaptation to drought and to contribute to an alleged inefficient
hydraulic system composed of tracheids (Bailey, 1953). Later,
Feild et al. (1998) proposed that the function of stomatal plugs
was to repel water lamina formation in leaves of Drimys winteri.
However, in contrast with D. winteri, our leaf hydrophobicity
results suggest that both sides of D. brasiliensis leaves are highly
wettable, and that the hydrophobicity of the abaxial leaf surfaces
decreases even more after being exposed to regular fog events
(Fig. 2). This reinforces the view that cuticles are complex and
dynamic structures that can modify their permeability in
response to changing environmental conditions (Schreiber et al.,
2001; Sch€onherr et al., 2005; Rosado et al., 2010). The difference
between leaf wettability on the adaxial and abaxial surfaces of
D. brasiliensis also suggests that the adaxial surface might contribute more to FWU, whereas the abaxial surface (where most
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(a)
(c)
(b)
(d)
Fig. 7 Changes in stem diameter (hst) (a), height (h) (b) and estimated total
leaf area (LA) (c) proportional to the initial values in control (n = 5), fog
(n = 6) and drought (n = 6) treatments at the end of the ecophysiological
experiment. Each box represents 50% of the observations; whiskers
represent the breadth of the distribution and the cross symbol represents
extreme values. The ‘molded waist’ portion of the box represents the range
of 95% around the median. (d) Drimys brasiliensis survival at the end of
the ecophysiological experiment. Bars represent the percentage of plants
alive in each treatment at the end of the experiment (Fogged, n = 12;
Control and Drought, n = 11). The same letters imply that the groups did
not differ significantly (Bonferroni test).
stomata are located), being more hydrophobic, might facilitate
gas exchange during and immediately after fog events.
FWU can occur in distinct plant taxa through a variety of leaf
mechanisms and structures, such as stomatal aperture (Eichert
et al., 2008; Burkhardt et al., 2012), guard cells (Schlegel et al.,
2005), trichomes (Schreiber et al., 2001; Sch€onherr, 2006),
hydathodes (Martin & von Willert, 2000) and, possibly, even by
stomatal plugs (Westhoff et al., 2009). FWU through the cuticle
can occur via polar pathways (aqueous pores; Sch€onherr, 1976;
Kerstiens, 2006) and ectodesmata (Franke, 1961; Sch€onherr,
2006). The existence of multiple ‘water entry pathways’ in leaves
of different plant taxa suggests that FWU might be selected for in
trees subjected to large soil-to-leaf water potential gradients; (e.g.
tall trees) and/or in environments in which leaf wetting events and
dry soils are common.
Fog internal hydraulic redistribution
We observed that fog water was not only absorbed by the leaves
of D. brasiliensis, but also internally redistributed through the
plant (Figs 4, 5). Reversal in stem sap flow during fog events has
been reported for two temperate gymnosperm species and for
Prosopis tamarugo in the Atacama desert (Sudzuki, 1969; Burgess
& Dawson, 2004; Nadezhdina et al., 2010), and, as far as we
know, this is the first evidence of fog hydraulic redistribution in
stems of a tropical cloud forest species. The observed sap flow
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reversal caused by FWU, not only in stems, but also in roots
(Fig. 4a), reinforces the idea that absorbed fog water can be transported to the soil by the plant. The deuterium enrichment of the
soil close to D. brasiliensis roots (Fig. 5) provides further evidence
that FWU-absorbed water is probably released into the rhizosphere, and not used solely to refill the plant’s internal capacitance, as observed for Sequoia sempervirens (Simonin et al., 2009).
However, as we did not sieve the soil samples before the analysis,
we cannot disregard the possibility that fine roots were present in
our soil samples. Hence, our isotope data do not provide definitive evidence that fog water was actually exuded from the roots to
the soil, and further experiments using root-excluding mesh
screens would be necessary to confirm this exudation. However,
considering that most plants do not have mechanisms that prevent water from flowing out of their roots towards the soil, at
least on a short time scale (Caldwell et al., 1998), and given the
existence of a water potential gradient sufficiently large to allow
water movement from leaves to the roots, it is likely that water
moved out of the roots into the soil.
The amount of water redistributed was proportionally higher
in the stems of D. brasiliensis when compared with other studies.
The reversal rates of sap flow peaked at c. 25% of maximum daily
transpiration rates in our glasshouse experiment, whereas it
peaked at 5–7% in S. sempervirens (Burgess & Dawson, 2004).
This difference in magnitude might even explain why fog decouples S. sempervirens water and carbon relations from soil water
deficit (Simonin et al., 2009), whereas, in D. brasiliensis, it slows
down the soil drying process (Fig. 6e).
In summary, our findings demonstrate a novel mechanism by
which plants use fog water; D. brasiliensis is not only able to
absorb water directly through its leaves (Figs 1, 3a), but can also
redistribute this water through its xylem (Fig. 4), rehydrating
plant tissues (Fig. 3b) and moving water to plant belowground
components (Fig. 5). This alternative water acquisition strategy
provided enough water to allow D. brasiliensis saplings to keep
the soil water content in their pots relatively stable for 2 months
without any additional water input (Fig. 6e), and allowed the
plants to keep a positive carbon gain and growth at higher levels
than the drought treatment (Figs 6, 7). We expect that FWU and
internal hydraulic redistribution might happen not only during
fog events, but also during any leaf wetting event that does not
significantly wet the soil, such as dew, light rain or drizzle.
Ecological importance
Drimys brasiliensis is very sensitive to soil drought and significantly reduced gas exchange, ΨPD, growth and survival in
response to decreases in soil water content (Figs 6, 7; Table 1).
By contrast, fog maintained the plant’s hydration (fog contributed as much as 42% of leaf water content) and allowed
D. brasiliensis to maintain its ecophysiological performance close
to the control treatment during the 2 months of the experiment.
The impact of nocturnal nebulizations on the soil water budget
of our glasshouse experiment may be explained by three mechanisms: (1) suppression of night-time transpiration during nebulization, which reduces the plant’s total water use; (2) fog internal
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hydraulic redistribution which rehydrates internal tissues
(Figs 3b, 4, 5) and might be used for transpiration, reducing the
uptake of soil water; and (3) fog hydraulic redistribution was
probably a consequence of the transport of fog water to the roots
(Figs 4, 5), such that the direct release of water into the soil could
also affect soil RWC.
There is some controversy about the consequences of wet
leaves on plant performance. We propose that, in some environments, the benefits to plant performance granted by FWU should
surpass the performance detriment caused by the reduction in gas
exchange when the leaf is wet. Considering that, during most leaf
wetting events (fog, drizzle and light rain), the photon flux density is greatly reduced, any reduction in gas exchange in leaves
caused by the formation of a water film during a foggy period
should not greatly reduce a plant’s total carbon balance. Thus,
FWU might provide an ecological advantage in environments in
which plant performance is limited by water, at least seasonally,
such as at our study site and in redwood forests in California,
where leaf water uptake is quite common (Limm et al., 2009). In
environments that are not limited by water during any season,
predictions about high leaf hydrophobicity in foggy environments (Smith & McClean, 1989) should still be the norm.
The ecological benefits granted by FWU are probably not limited to the effects shown in our study (maintenance of gas
exchange, growth and survival; Figs 6, 7). Considering that the
water absorbed by FWU reaches plant’s roots and also probably
their rhizosphere, all the effects associated with the moistening of
roots by hydraulic redistribution could also be happening here,
including the minimization of root embolism and prolongation
of root life span (Domec et al., 2004, 2006; Bauerle et al., 2008),
benefits to rhizosphere fungal associations (Querejeta et al.,
2007) and even increasing nutrient availability (Dawson, 1997;
Pang et al., 2013).
As our experiment was performed on young isolated plants
under glasshouse conditions, our results on the effects of FWU
on plant performance might be hard to extrapolate to adult individuals under field conditions. However, drought can be especially detrimental to younger plants, because of the smaller
volume of soil explored by their roots. Therefore, FWU probably
plays an important role in seedling establishment and survival
during seasonal droughts.
Conclusions
In our study, we have shown that FWU promotes fog water redistribution, a process that plays an important role in plant survival
and growth during low rainfall periods in tropical environments.
Our results strengthen the soil–plant–atmosphere continuum
(SPAC) view proposed by Simonin et al. (2009) that water movement in plants should be seen as a true continuum between all
potential water sources. In our case, the establishment of a water
potential gradient between wet leaves and dry soil was enough to
induce a basipetal sap flow in D. brasiliensis.
Considering that FWU mitigates the deleterious effects of
drought in plant performance, such as carbon starvation and
hydraulic failure (McDowell et al., 2008), we believe that FWU of
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fog water might be a key trait explaining the ecological distribution of D. brasiliensis in high-altitude environments that are often
subjected to seasonal droughts (Bertoncello et al., 2011). Global
climatic models predict an increase in the average height of cloud
formation in tropical cloud forests (Still et al., 1999), leading to a
drier montane climate with fewer fog events. Atlantic cloud forests
may experience a change in their composition and functioning in
the future, because of the predicted higher mortality of fog-dependent species. More detailed studies on the relationships between
fog, drought and vegetation functioning are necessary to better
understand and predict the extent of these changes.
Our study, together with several others that have recently challenged the unidirectional SPAC model (Burgess & Dawson,
2004; Oliveira et al., 2005; Simonin et al., 2009; Nadezhdina
et al., 2010), urges new parameterization of ecosystems and
global climate models that consider soil water as the only water
source used by vegetation for carbon fixation and evapotranspiration. Considering D. brasiliensis as a model tree for tropical cloud
forests, and assuming that FWU and fog hydraulic redistribution
are common physiological processes in these forests, we can
assume that they play an important role in the hydrology and
productivity of these ecosystems, just like hydraulic redistribution
between soil layers in lowland tropical forests (Ryel et al., 2002;
Lee et al., 2005).
Acknowledgements
The authors gratefully acknowledge the following: financial
support by S~ao Paulo Research Foundation (FAPESP) (Grant
number 10/17204-0 to R.S.O. and Biota Gradiente Funcional
03/12595-7); National Council for Scientific and Technological
Development (CNPq) and Higher Education Co-ordination
Agency (CAPES) (scholarships to A.L.L. and C.B.E.); Graduate
Program in Ecology and Plant Biology from University of
Campinas (UNICAMP), Forestry Institute (COTEC -IF); staff of
the CJSP and Umberto Bonini for logistic support; research support and facilities generously offered by the Plant Anatomy and
Physiology Laboratories of UNICAMP (Profs. Sandra Guerreiro,
Marilia Castro, Paulo Mazzafera and their students) and Federal
University of Rio Grande do Sul (UFRGS) (Prof. Jorge Mariath,
Alexandra Mastroberti and Carlos Widholzer); Isotope Ecology of
Center for Nuclear Energy in Agriculture (CENA) (Prof. Plinio
Camargo, Marcelo Moreira, Luiz Martinelli, Geraldo Arruda and
Maria Antonia Perez); Leonardo Jorge for assistance with analysis;
Stephen Burgess for helping us with the sap flow data; Graham
Zemunik and Hans Lambers for providing comments on the
manuscript.
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