Thresholds for leaf damage due to dehydration: declines of hydraulic function, stomatal conductance and cellular integrity precede those for photochemistry

Abstract

Given increasing water deficits across numerous ecosystems world‐wide, it is urgent to understand the sequence of failure of leaf function during dehydration.
We assessed dehydration‐induced losses of rehydration capacity and maximum quantum yield of the photosystem II (Fv/Fm) in the leaves of 10 diverse angiosperm species, and tested when these occurred relative to turgor loss, declines of stomatal conductance gs, and hydraulic conductance Kleaf, including xylem and outside xylem pathways for the same study plants. We resolved the sequences of relative water content and leaf water potential Ψleaf thresholds of functional impairment.
On average, losses of leaf rehydration capacity occurred at dehydration beyond 50% declines of gs, Kleaf and turgor loss point. Losses of Fv/Fm occurred after much stronger dehydration and were not recovered with leaf rehydration. Across species, tissue dehydration thresholds were intercorrelated, suggesting trait co‐selection. Thresholds for each type of functional decline were much less variable across species in terms of relative water content than Ψleaf.
The stomatal and leaf hydraulic systems show early functional declines before cell integrity is lost. Substantial damage to the photochemical apparatus occurs at extreme dehydration, after complete stomatal closure, and seems to be irreversible.

Full text

Thresholds for leaf damage due to dehydration: declines of
hydraulic function, stomatal conductance and cellular integrity
precede those for photochemistry
Santiago Trueba
1
, Ruihua Pan
1,2
, Christine Scoffoni
1,3
, Grace P. John
1,4
, Stephen D. Davis
5
and Lawren
Sack
1
1
Department of Ecology and Evolutionary Biology, University of California Los Angeles, 621 Charles E. Young Drive South, Los Angeles, CA 90095, USA;
2
School of Ecology and
Environment, Inner Mongolia University, 235 University West Road, Hohhot, Inner Mongolia 010021, China;
3
Department of Biological Sciences, California State University Los Angeles,
5151 State University Drive, Los Angeles, CA 90032, USA;
4
Department of Integrative Biology, University of Texas at Austin, 2415 Speedway, Austin, TX 78712, USA;
5
Natural Science
Division, Pepperdine University, Malibu, CA 90263-4321, USA
Author for correspondence:
Santiago Trueba
Tel: +1 310 465 8307
Email: strueba@gmail.com
Received: 6 October 2018
Accepted: 18 February 2019
New Phytologist (2019) 223: 134–149
doi: 10.1111/nph.15779
Key words: drought stress, leaf hydraulics,
photosynthesis, recovery, rehydration,
stomatal conductance, turgor loss point,
vulnerability.
Summary
Given increasing water deficits across numerous ecosystems world-wide, it is urgent to
understand the sequence of failure of leaf function during dehydration.
We assessed dehydration-induced losses of rehydration capacity and maximum quantum
yield of the photosystem II (F
v
/F
m
) in the leaves of 10 diverse angiosperm species, and tested
when these occurred relative to turgor loss, declines of stomatal conductance g
s
, and
hydraulic conductance K
leaf
, including xylem and outside xylem pathways for the same study
plants. We resolved the sequences of relative water content and leaf water potential Ψ
leaf
thresholds of functional impairment.
On average, losses of leaf rehydration capacity occurred at dehydration beyond 50% decli-
nes of g
s
,K
leaf
and turgor loss point. Losses of F
v
/F
m
occurred after much stronger dehydra-
tion and were not recovered with leaf rehydration. Across species, tissue dehydration
thresholds were intercorrelated, suggesting trait co-selection. Thresholds for each type of
functional decline were much less variable across species in terms of relative water content
than Ψ
leaf
.
The stomatal and leaf hydraulic systems show early functional declines before cell integrity
is lost. Substantial damage to the photochemical apparatus occurs at extreme dehydration,
after complete stomatal closure, and seems to be irreversible.
Introduction
The increasing frequency and severity of declines in precipitation
result in critical drought events and hydrological imbalances
(Trenberth et al., 2014), inducing tree mortality and shifting
species distributions across global ecosystems (Breshears et al.,
2005; Allen et al., 2010, 2015). The impacts of drought over
large surfaces in crops and natural ecosystems can be evaluated
using remotely sensed estimation of canopy water content based
on light absorption assessed from hyperspectral measurements
(Asner et al., 2016). Yet, such impacts may represent extreme
dehydration, beyond the point at which major functions have
declined. Indeed, there has been fragmentary knowledge of the
sequence of dehydration-induced reduction of stomatal conduc-
tance, leaf hydraulic transport, cellular integrity, and chloroplast
function. Clarifying thresholds in key physiological functions can
thus be important for inferring leaf-level mechanisms, the signifi-
cance of declines inferred from remotely sensed variables, and for
the improvement of ecosystem-wide models (Adams et al.,
2017). A recent meta-analysis has refined hypotheses for how
plant organs differ in their water status thresholds for dysfunction
under drought stress (Bartlett et al., 2016). However, extensive
measurements on a given set of species are necessary to test that
sequence, and especially to resolve the placement of irreversible
leaf damage. We aimed to integrate key stomatal, photosyn-
thetic, hydraulic, turgor loss, and damage processes into a uni-
fied sequence of leaf response to drought for 10 diverse
angiosperm species.
Most leaf water exchange occurs through the stomatal pores.
Stomatal sensitivity to dehydration can be assessed by measuring
the behavior of stomatal conductance g
s
(see Table 1 for a list of
abbreviations and units) under decreasing water status (Klein,
2014). Declines of g
s
with declining values of total relative water
content (RWC; combining apoplastic and symplastic water con-
tents) and leaf water potential Ψ
leaf
, are expected to reduce water
loss and to avoid reaching dangerous xylem tensions (Meinzer
134 New Phytologist (2019) 223: 134–149 Ó2019 The Authors
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et al., 2009). Other traits indicating drought-induced hydraulic
dysfunction include the water status at wilting or turgor loss
point (TLP; Bartlett et al., 2012a) and the decline of leaf lamina
hydraulic conductance K
leaf
, a major determinant of plant
drought responses and ecological preferences (Blackman et al.,
2009; Nardini & Luglio, 2014; Scoffoni & Sack, 2017). A key
impact of outside-xylem hydraulic conductance K
ox
in driving
whole-leaf hydraulic decline with dehydration has been recently
emphasized (Scoffoni et al., 2017). Recent work has suggested
that xylem-specific hydraulic decline K
x
occurs only after stom-
atal closure (Hochberg et al., 2017; Scoffoni & Sack, 2017; Skel-
ton et al., 2017a), and a meta-analysis found that most hydraulic
traits conferring drought tolerance are correlated (Bartlett et al.,
2016). Yet, the Ψ
leaf
and RWC thresholds inducing TLP, g
s
,
K
leaf,
K
ox
, and K
x
declines have not been integrated and directly
compared experimentally.
Several studies have shown that stomatal pores reopen after
plant rehydration, and reopening is associated with the recovery
of K
leaf
and gas exchange (Miyashita et al., 2005; Blackman et al.,
2009; Brodribb & Cochard, 2009; Martorell et al., 2014; Cai
et al., 2015; Li et al., 2016; Skelton et al., 2017b). However, in
contrast to g
s
, there may be limited recovery of photochemistry
and mesophyll conductance after rehydration, which constrain
full recovery of overall photosynthetic rate (Miyashita et al.,
2005; Galmes et al., 2007). The decline of leaf photochemical
performance under water deficit can be estimated by the measure-
ment of Chl fluorescence (ChlF) (Baker, 2008). Among the ChlF
parameters, the decline of the maximum quantum efficiency of
photosystem II (PSII) photochemistry (F
v
/F
m
) represents the
light-harvesting function of the chloroplast (Murchie & Lawson,
2013), and is a parameter that can be used as an index of
drought-induced injury in leaves (Guadagno et al., 2017). Previ-
ous studies have reported that that PSII photochemistry was little
affected by decreasing RWC between 100% and 50% (Genty
et al., 1987; Lawlor & Cornic, 2002). Dehydration causes
changes in cell volume and osmotic concentration that can lead
to significant structural damage; therefore, we analyzed the
percentage loss of F
v
/F
m
(PLCF) as a proxy of dehydration-
induced chloroplast dysfunction within the sequence of
dehydration effects.
Under strong dehydration, vegetative tissues may experience
irreversible cellular damage. Irreversible mesophyll damage has
been assessed by dehydrating tissues and testing their ability to
rehydrate (i.e. their loss of rehydration capacity). Irreversible
injury in evergreen Mediterranean leaves has been proposed to
occur at a percentage loss of rehydration capacity (PLRC) of
≥10% (Oppenheimer, 1963; Oppenheimer & Leshem, 1966).
The rehydration technique to estimate loss of viability was
recently refined and applied to 18 southern California species,
showing that water status thresholds for PLRC are related to
other drought tolerance indicators and leaf structural traits such
as TLP and leaf mass per area (LMA; John et al., 2018). More-
over, thresholds for PLRC varied across biomes, suggesting that
leaf rehydration capacity may predict drought tolerance across
species (John et al., 2018).
Dehydration-response thresholds can be expressed in terms of
declining RWC or Ψ
leaf
. Which of these water status variables is
more directly sensed by leaf cells, and more active in triggering
dehydration-induced responses, has been controversial. Whereas
Ψ
leaf
declines reflect xylem tensions that would trigger air seeding
and reduction of K
x
, there is no clear understanding yet of the
direct mechanisms by which low leaf water status drives declines
of stomatal closure, K
ox
and K
leaf
, along with PLRC or PLCF,
and thus it is uncertain whether RWC or Ψ
leaf
would be most
meaningful (Sack et al., 2018). Indeed, some have suggested that
RWC might better represent the water status that is sensed by
cells experiencing dehydration (Sinclair & Ludlow, 1985), espe-
cially as the decline of RWC appears to be critical in the accumu-
lation of the hormone abscisic acid (ABA), consequently driving
important physiological responses such as stomatal closure (Bro-
dribb & McAdam, 2011; Sack et al., 2018). Further, species that
diverge in thresholds of dehydration responses in terms of Ψ
leaf
may show convergence in thresholds in terms of RWC (Sack
et al., 2018). The incorporation of drivers of physiological
responses to dehydration, whether expressed in terms of Ψor
RWC, into predictive models is critical to understand and predict
ecosystem responses to drought (Anderegg et al., 2017).
We analyzed the dehydration-induced loss of leaf rehydration
capacity, and F
v
/F
m
along with stomatal conductance and other
indices of leaf dehydration tolerance previously measured on the
same individuals, for 10 species with diverse sensitivities to water
deficit. Using these leaf dehydration responses, we tested key
hypotheses: (1) the declines of leaf rehydration capacity and pho-
tochemistry vary across species and occur after stomatal closure
and decline of leaf hydraulic conductance; (2) F
v
/F
m
may not
recover after rehydration due to permanent dehydration-induced
damage; (3) across-species, water status thresholds for loss of
stomatal, hydraulic, and photochemical function and cell
integrity will be correlated; (4) thresholds in terms of Ψ
leaf
will be
more variable across species than in terms of RWC. Addressing
these hypotheses resulted in a comprehensive ‘timeline’ of leaf
Table 1 Symbols and definitions of water status indices and thresholds
measured.
Symbol Definition Units
RWC Relative water content %
Ψ
leaf
Leaf water potential MPa
F
v
/F
m
Maximum quantum efficiency
of photosystem II photochemistry
Unitless
K
leaf
Leaf hydraulic conductance mol m
2
s
1
MPa
1
K
ox
Outside-xylem leaf hydraulic
conductance
mol m
2
s
1
MPa
1
K
x
Xylem vein leaf hydraulic
conductance
mol m
2
s
1
MPa
1
g
s
Stomatal conductance mmol m
2
s
1
PLRC Percentage loss of rehydration
capacity
%
PLCF Percentage loss of Chl
fluorescence (F
v
/F
m
)
%
TLP Turgor loss point MPa
LA Leaf area cm
2
LMA Leaf mass per area g m
2
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functional impairment with decreasing leaf water status during
drought stress.
Materials and Methods
Plant material
We studied 10 angiosperm species varying in leaf hydraulic vul-
nerability and drought tolerance (Table 2; Guyot et al., 2012;
Scoffoni et al., 2017, 2011). Measurements were made from
December 2016 to April 2017 on three mature individuals per
species. Individuals are located in the campus of University of
California Los Angeles and Will Rogers State Park, Los Angeles,
California. Sun-exposed shoots 20–65 cm in length were col-
lected early in the morning, to avoid native embolisms, and
immediately transported to the laboratory in dark plastic bags
containing moist paper towels, recut underwater by three nodes,
placed with cut ends under water, and rehydrated overnight
covered in plastic for >12 h. Thirteen fully expanded leaves per
individual were excised from the shoots, avoiding leaves with
pathogen or herbivore damage. Previous studies have shown
similar hydraulic declines under dehydration alternatively using
bench-dried shoots or droughted plants (Blackman et al., 2009;
Pasquet-Kok et al., 2010). We therefore used a bench-drying
approach to assess dehydration impacts on hydraulic, rehydra-
tion, and photochemistry capacities. To isolate the effect of water
scarcity on leaf function, dehydration for all measurements was
carried out at room temperature and low irradiance levels, with a
range in photosynthetic photon flux density of 4–19 lmol
m
2
s
1
, and temperature and relative humidity ranges of 20–
25°C and 35–45%, respectively.
Determination of relative water content and water status
thresholds for loss of rehydration capacity
Fully turgid leaves were weighed immediately after excision
using an analytical balance (0.01 mg; AB265-S; Mettler
Toledo, Greifensee, Switzerland). ChlF was measured promptly
after weighing, as described in the following section. The pro-
jected leaf area (LA, cm
2
) was measured with a leaf area meter
(LI-3100; Li-Cor Biosciences, Lincoln, NE, USA) or scanned
with a flatbed scanner (Canon Scan Lide 90; Canon, New
York, NY, USA) and measured using IMAGEJ v.1.51h (NIH
Image; Bethesda, MD, USA). Leaves were then placed over a
bench fan to dehydrate under ambient light. Three randomly
selected leaves were removed from the drying bench at differ-
ent time points to represent a gradual dehydration gradient
and measured for LA and ChlF. Species varied in the times
necessary for dehydration depending on their water loss rates;
for instance, the thin leaves of Lantana camara and Salvia
canariensis required only 24 h to reach very low water contents,
whereas Magnolia grandiflora required up to 10 d. Dehydrated
leaves were placed in water-filled 15 ml polypropylene tubes
(Fisher brand; Fisher Scientific Co., Waltham, MA, USA),
covered, and rehydrated overnight. Only the petioles of leaves
were immersed under water, avoiding contact of the leaf blades
with water, for 10–12 h rehydration, longer than the minimum
of 8 h of rehydration required (John et al., 2018). LA, mass,
and ChlF were then measured on the rehydrated leaves.
Finally, leaves were oven dried at 70°C for 72 h and leaf dry
mass was determined. LMA (g m
2
) was determined by divid-
ing the dry leaf mass by the area of the turgid leaf.
We calculated the saturated water content (SWC, g g
1
) of sat-
urated (i.e. fully turgid before dehydration) and rehydrated leaves
as:
SWCs¼MsMd
Md
Eqn 1
SWCr¼MrMd
Md
Eqn 2
where M
d
,M
s
, and M
r
are the mass values (in grams) of dry, satu-
rated (i.e. fully turgid), and rehydrated leaves, respectively. The
SWC of rehydrated leaves and saturated leaves were used to cal-
culate the percentage loss of rehydration capacity (PLRC, %) as:
Table 2 List of measured angiosperm species, values of leaf structural variables, and thresholds of dehydration tolerance.
Species Family Native biome and origin Leaf area (cm
2
)
Leaf mass
per area (g m
2
)
RWC (%) at:
PLRC
10
PLRC
50
PLCF
10
PLCF
50
Cercocarpus betuloides Rosaceae Mediterranean, North America 8 0.31 314 12 58.97 36.86 65.26 4.94
Comarostaphylis
diversifolia
Ericaceae Mediterranean, North America 9 0.53 306 8 57.27 35.61 40.38 26.46
Hedera canariensis Araliaceae Temperate forest, Africa 44 3.60 71 3 78.42 45.47 25.27 8.81
Heteromeles arbutifolia Rosaceae Mediterranean, North America 18 1.22 138 3 67.92 36.78 28.52 15.02
Lantana camara Verbenaceae Tropical dry forest, pantropical 15 0.68 65 2 63.81 34.18 30.18 8.98
Magnolia grandiflora Magnoliaceae Temperate forest, North America 45 2.18 256 4 83.05 43.29 99.55 28.58
Malosma laurina Anacardiaceae Mediterranean, North America 14 0.83 189 3 83.75 43.51 45.75 17.63
Quercus agrifolia Fagaceae Mediterranean, North America 9 0.55 205 7 79.60 43.56 43.27 14.69
Rhaphiolepis indica Rosaceae Temperate forest, Asia 10 0.41 217 3 62.82 40.27 32.77 14.69
Salvia canariensis Lamiaceae Temperate forest, Africa 37 3.69 79 2 66.52 41.75 24.97 8.09
Mean SE of leaf structural variables (leaf area, leaf mass per area) are provided. PLCF, percentage loss of Chl fluorescence; PLRC, percentage loss of
rehydration capacity; RWC, relative water content.
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136

PLRC ¼100 1SWCr
SWCs
 Eqn 3
The total relative water content (RWC, %) of dehydrated
leaves was calculated as:
RWC ¼100 Mde Md
MsMd
 Eqn 4
where M
de
,M
d
, and M
s
are the mass values (in grams) of dehy-
drated, dry, and water saturated leaves, respectively. Rehydration
curves for each species were determined by fitting functions to
the increase of PLRC with decreasing RWC, as described in the
Data analyses section. The RWC at which the 10%, 20%, and
50% of rehydration capacity were lost (RWC at PLRC
10
,
PLRC
20
, and PLRC
50
, respectively) were extracted from the
rehydration curves.
Maximum quantum yield of PSII and water status
thresholds for loss of ChlF
ChlF was measured using a portable pulse-modulated fluorome-
ter (OS1p; Opti-Sciences, Hudson, NH, USA) with a modulated
light source of 0.2 lmol m
2
s
1
at 660 nm and a saturation
pulse from a white light-emitting diode with an intensity of
7700 lmol m
2
s
1
for a duration of 1.5 s. Measurements were
performed on the adaxial surface at the middle zone of the leaf
blade, avoiding main veins. Leaves were dark adapted for 30 min
before measurements using leaf clips to avoid the effects of non-
photochemical acute photoinhibition during measurements. The
maximum quantum yield of the PSII was estimated as the ratio
of variable to maximum fluorescence:
Fv
Fm
¼FmFo
Fm
Eqn 5
where F
m
and F
o
are respectively the maximal and minimum flu-
orescence. F
v
/F
m
indicates the maximum efficiency at which light
absorbed by the PSII for reduction of the primary electron accep-
tor quinone molecule of PSII (Genty et al., 1989). In this study
we assessed the percentage loss of ChlF (PLCF, %) as an index of
loss of fluorescence capacity after dehydration, using the ratio of
F
v
/F
m
of individual leaves at dehydration, and at saturated status:
PLCF ¼100 1Fv=Fm dehydrated
Fv=Fm saturated
Eqn 6
We then analyzed the variation of PLCF as a function of
RWC to estimate the RWC corresponding to 10%, 20%, and
50% loss in F
v
/F
m
. We tested F
v
/F
m
before and after leaf rehy-
dration to determine the recoverability of F
v
/F
m
. To analyze
the recovery of F
v
/F
m
, we considered four stages of water stress
based on the minimum RWC reached during dehydration:
operational (stage 1; 100–90% RWC), corresponding to leaves
with RWC values above TLP; mild (stage 2; 90–70% RWC);
moderate (stage 3; 70–40% RWC); and severe (stage 4; 40–
0% RWC).
Given declines in F
v
/F
m
during dehydration, we tested the pos-
sibility that these arose due to Chl degradation in excited leaves.
We measured Chl content during dehydration for a subset of
four species with different drought tolerances, Cercocarpus
betuloides,Comarostaphylis diversifolia,L. camara, and Malosma
laurina. Chl concentration of dehydrated leaves was measured
using a portable Chl meter (SPAD-502; Minolta Camera Co.,
Osaka, Japan); (Uddling et al., 2007). Values for Chl concentra-
tion were also corrected for the effects of leaf area shrinkage dur-
ing dehydration by multiplying the values by the ratio of
dehydrated leaf area/saturated leaf area.
The test of the decline of F
v
/F
m
during rehydration was con-
ducted for all 10 species under the ambient light of the laboratory
(7 2lmol m
2
s
1
) specifically to isolate the effect of dehydra-
tion on leaf photochemistry without excess light and temperature
stresses. However, we further tested the effect of dehydration on
F
v
/F
m
under high irradiance for the subset of four species con-
trasting in drought tolerances, C. betuloides,C. diversifolia,
L. camara, and M. laurina. Twenty-five leaves per species were
dehydrated under a light source with an irradiance of
343 42 lmol m
2
s
1
, and we measured F
v
/F
m
using the previ-
ously described protocol. We estimated PLCF thresholds of
leaves dehydrated under high irradiance and compared these
thresholds with those measured under low irradiance.
Stomatal conductance and water status thresholds for
stomatal closure
The water status values inducing 20%, 50%, and 80% declines
in stomatal conductance g
s
were determined for the same individ-
ual plants using shoot drying experiments. We measured g
s
on
the abaxial surface of leaves using a porometer (AP-4; Delta-T
Devices Ltd, Cambridge, UK) during progressive dehydration.
After porometer measurements, leaves were placed in bags and
allowed to equilibrate for a minimum of 30 min before measure-
ment of Ψ
leaf
using a pressure chamber (Plant Moisture Stress
pressure chamber model 1000; PMS Instrument Co., Albany,
OR, USA).
Additional hydraulic data used to determine the sequence
of functional loss during dehydration
We synthesized the data from previous studies on the same indi-
vidual plants of the same species for TLP measured using the
osmometric method (Bartlett et al., 2012b) and thresholds for
leaf hydraulic vulnerability (Scoffoni et al., 2011; Guyot et al.,
2012). Additionally, we gathered previously published data of
the thresholds for the declines of xylem and outside-xylem
hydraulic conductance (K
x
and K
ox
, respectively) for eight of the
species studied (Scoffoni et al., 2017). Although interindividual
plasticity was eliminated by sampling the exact same individuals,
interannual plasticity would introduce a level of uncertainty in
the comparison of thresholds. However, such plasticity is not
expected to reduce the robustness in our comparison of
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Phytologist Research 137

thresholds. Interannual comparisons of TLP measurements of
the individuals studied showed high similarity in TLP values
from different years (r
2
=0.88, for 2012 vs 2016 measurements;
n=13; L. Sack, unpublished data). Moreover, previous studies
showed similar K
leaf
vulnerability for at least five species measured
in different years (Brodribb & Holbrook, 2003, 2006; Scoffoni
et al., 2011; Guyot et al., 2012; L. Sack, unpublished data).
Pressure–volume curves and conversion of water status
indices
We estimated dehydration-induced thresholds of leaf damage in
terms of both Ψ
leaf
and RWC. The conversion of Ψ
leaf
to RWC
was employed to determine the RWC thresholds of stomatal con-
ductance and the previously published dehydration tolerance
traits (TLP, K
leaf
,K
ox
, and K
x
). Similarly, we converted the RWC
thresholds of PLRC and PLCF measured in this study into Ψ
leaf
thresholds. For these conversions, we used equations based on
pressure–volume curve parameters determined for leaves of the
same individual plants using the bench-dry method (Sack & Pas-
quet-Kok, 2011; Sack et al., 2018). Five leaves per species were
measured to determine the relation of Ψ
leaf
and mass during
dehydration. From these curves we determined osmotic potential
at turgor loss point p
tlp
and at full turgor p
o
, RWC at turgor loss
point RWC
tlp
, and modulus of elasticity e. Pressure–volume
curve parameters for each species studied are available in the Sup-
porting Information Table S1. Notably, extremely low RWC
thresholds (i.e. <40%) could not be converted into Ψ
leaf
thresh-
olds using these equations, as this required extrapolation beyond
the pressure–volume curve data, which led to unreliable esti-
mates, especially given uncertainty in the estimation of apoplastic
fraction (Arndt et al., 2015). Therefore, WPLCF10 was only com-
puted for five species, and WPLCF50 only for L. camara.WPLCF50
could be calculated for only three species, and thus these thresh-
olds were not included in further analyses.
Data analyses
The loss of rehydration capacity and ChlF as a function of leaf
relative water content was determined by fitting the relationship
of the dependent variables PLRC and PLCF against RWC using
three differently shaped functions: linear, y=a+bx; exponential,
y=aexp(bx); and sigmoidal, y¼a=f1þexp½ðxXoÞ=bg.
Functions were fitted, parameterized and compared using maxi-
mum likelihood, and for each species’ response the function was
selected according to the lowest Akaike information criterion
corrected for small n(Burnham & Anderson, 2003). The
selected functions were used to estimate 10%, 20%, and 50%
losses in rehydration capacity and ChlF for each species. The
same procedure was used to estimate Ψ
leaf
thresholds inducing
stomatal closure.
We built a linear-mixed effect model to compare F
v
/F
m
values
in dehydrated and rehydrated leaves with those of water-saturated
leaves using the R packages LME4 (Bates et al., 2014) and
LMERTEST (Kuznetsova et al., 2017). Hydration treatments were
included as fixed effects, whereas species and individuals were
included as random effects to account for repeated measures
within and across species. Comparisons across hydration stages
were assessed using t-tests based on Satterhwaite’s degrees of free-
dom method (Kuznetsova et al., 2017). Additionally, differences
in F
v
/F
m
among saturated leaves, leaves dehydrated to different
stages of water stress, and rehydrated leaves were assessed using a
one-way ANOVA with post-hoc Tukey’s honest significant dif-
ference using 95% confidence intervals. The same analysis was
applied to assess differences in Chl concentration in leaves at dif-
ferent stages of water stress. Differences of F
v
/F
m
declines of
leaves under high vs low irradiance were assessed using a paired t-
test across the four species tested.
We used both indices of water status, RWC and Ψ
leaf
,to
assemble sequences of thresholds of functional decline and to
compare the variability of thresholds among both water status
indices. To establish the sequence of drought response thresholds,
we used t-tests to compare species means for RWC and Ψ
leaf
thresholds of drought responses. Ψ
leaf
values were multiplied by
1 to generate positive values for analyses. We assessed the vari-
ability of RWC and Ψ
leaf
thresholds overall by comparing their
coefficients of variation (CVs). CV equality was tested using the
modified signed-likelihood ratio test (Krishnamoorthy & Lee,
2014), which was implemented using the R package CVEQUALITY
(Marwick & Krishnamoorthy, 2016).
We estimated trait–trait relationships across species using cor-
relation analyses on mean species trait values. Correlations were
evaluated for both untransformed data and log-transformed data
to test approximately linear or curvilinear (i.e. power-law) rela-
tionships. When relationships were found between two ‘depen-
dent’ variables, we fitted standardized major axes to plot these
relationships using the R package SMATR (Warton et al., 2012).
Our dataset is available in Table S2. All data analyses were carried
out using R v.3.4.1 (R Core Team, 2017).
Results
Loss of rehydration capacity with leaf dehydration
Declining RWC strongly reduced leaf rehydration capacity in
the 10 study species (Fig. 1). The sigmoid function was selected
by maximum likelihood for the relation of PLRC and RWC for
most species analyzed, whereas this relation was best explained by
a linear function only for M. grandiflora (adjusted r
2
of the
selected models ranged from 0.93 to 0.98; Fig. 1; Table S3).
Most species had relatively slight losses (<10%) of rehydration
capacity until reaching c. 70% RWC (Fig. 1), with subsequently
strong decreases until reaching a plateau at RWC of c. 25%
(Fig. 1), after which the loss in rehydration capacity became
weaker as RWC approached zero. Across species, the mean
RWC values inducing a 10%, 20%, and 50% loss in rehydration
capacity were 70 3%, 60 3%, and 40 1%, respectively
(mean SE; Table2). The minimum and maximum RWCPLRC50
were 34% and 45%, for L. camara and Hedera canariensis,respec-
tively (Fig.1d,e). Indices of leaf rehydration capacity were interre-
lated: RWC
PLRC50
was strongly correlated with RWCPLRC10 and
RWCPLRC20 (r=0.82–0.85; P=0.002–0.003). Across species,
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(a) (b)
(c) (d)
(e) (f)
(g) (h)
(i) (j)
Fig. 1 Response of percentage loss of
rehydration capacity (PLRC) over decreasing
relative water content (RWC) for the 10
species studied. Gray points are measured
leaves, and solid regression lines are best-fit
models. The dashed lines in each plot
indicate the RWC associated with 10%
(blue) and 50% (red) losses of rehydration
capacity. ***,P≤0.001. (a–j) refer to
different species in the panel.
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mean WPLRC10 was 2.71 0.33, and mean WPLRC20 was
4.64 0.89, and Ψ
leaf
thresholds of leaf rehydration were corre-
lated (r=0.77; P=0.04). The RWC and Ψ
leaf
thresholds of rehy-
dration capacity were statistically independent of LA (RWCPLRC50:
r=0.56; P=0.09; and WPLRC10 :r=0.15; P=0.74) and LMA
(RWCPLRC50:r=0.17; P=0.64; and WPLRC10:r=0.53;
P=0.22).
Response of PSII yield to dehydration and rehydration
F
v
/F
m
declined strongly with dehydration to very low RWC
(Fig. 2). In most species, percentage losses of F
v
/F
m
increased
exponentially with decreasing RWC (Fig. 2), whereas a sigmoid
model was selected for C. diversifolia (Fig. 2b), Heteromeles
arbutifolia (Fig. 2c), and Quercus agrifolia (Fig. 2h) (adjusted r
2
ranged from 0.47 to 0.97; Table S4). During the first phases of
leaf dehydration, F
v
/F
m
remained unresponsive to declining
RWC (Fig. 2), and in most species the decline in F
v
/F
m
became
substantial only after reaching RWC of c. 40%, with dramatic
reductions near RWC of 20%. The mean RWC values associated
with 10%, 20%, and 50% losses in F
v
/F
m
were 44 7%, 32 5-
%, and 16 3%, respectively (Table 2). Cercocarpus betuloides
and M. grandiflora were the species with the most and least
desiccation-resistant photosynthetic light reaction apparatus,
with RWCPLCF50 ¼5%and RWCPLCF50 ¼29%, respectively
(Fig. 2a,f).
Rehydrated leaves had significantly lower F
v
/F
m
and did not
recover to their initial values (Table S5). F
v
/F
m
varied signifi-
cantly across levels of water stress (Fig. 3; ANOVA, P<0.05).
However, F
v
/F
m
did not recover significantly at any level of water
stress (Fig. 3). RWC thresholds for PLCF correlated positively
with LMA across species, such that species with higher LMA also
had a more sensitive photosynthetic light reaction apparatus
(r=0.64; P=0.048 for RWCPLCF10 ;r=0.67; P=0.033 for
RWCPLCF20 ). RWC thresholds for PLCF were statistically inde-
pendent of LA. The WPLCF10 could be extrapolated only for five
species and was 15.2 5.14 MPa on average.
For four species tested for potential Chl concentration decline
in dehydrating leaves, we found instead an increase in Chl concen-
tration with dehydration (Fig. S1a). However, after correction for
the area shrinkage of leaves during dehydration, we found that Chl
concentrations remained constant during dehydration (Fig. S1b).
We tested the influence of high irradiance (343 
42 lmol m
2
s
1
) vs ambient laboratory irradiance (7 
2lmol m
2
s
1
) on the decline of F
v
/F
m
with dehydration in
four species (Fig. 4; Table S4). Although there was an empirical
trend for earlier F
v
/F
m
decline during dehydration under high
irradiance (Fig. 4), we found no statistical differences in the
RWC thresholds of F
v
/F
m
decline with irradiance.
Sequence of leaf functional failure during dehydration, and
comparisons of threshold variability in terms of RWC and
Ψ
leaf
We built a sequence of dehydration-induced leaf functional
decline as defined by RWC thresholds (Fig. 5). The 20% declines
in leaf hydraulic conductance K
leaf 20
and in stomatal conduc-
tance g
s20
occurred at similar RWC thresholds (Fig. 5;
Table S6). The 50% decline of outside-xylem leaf conductance
K
ox 50
was followed sequentially by those of g
s50
and K
leaf 50
,
which overlapped across the measured species (Fig. 5; Table S6).
TLP occurred after the 50% decline of most of the hydraulic
traits included in the analysis (Fig. 5; Table S6). However, TLP
occurred before substantial limitations in stomatal and leaf
hydraulic conductance (Fig. 5). The average thresholds for 80%
declines in stomatal conductance overlapped with TLP and with
K
leaf 80
(Fig. 5; Table S6), and was followed sequentially by
those of K
ox 88
,K
x50
, PLRC
10
,K
x88
, PLCF
10
, PLRC
50
, and
PLCF
50
.
Declines in rehydration capacity occurred late in the sequence
of functional decline (Fig. 5). Most of the declines in hydraulic
conductance and stomatal activity occurred before PLRC
10
, with
only K
x88
following in the dehydration-induced injury sequence
(Fig. 5), and both traits shared similar RWC thresholds on aver-
age across species (Table S6). Finally, declines in F
v
/F
m
took
place at the end of the leaf injury sequence, under high or low
irradiance, with PLCF
10
and PLCF
50
occurring at very low
RWC (Figs 4, 5), and after a substantial decline of the other
dehydration tolerance traits considered. Overall, the sequence of
dehydration-induced leaf functional impairment was similar
across species (Fig. S2) with some differences observed, especially
at the first stages of dehydration. In general, Ψ
leaf
thresholds of
leaf functional decline occurred in a sequence similar to those
estimated using RWC (Fig. 6; Table S7). All the Ψ
leaf
thresholds
of dehydration responses analyzed had significantly greater vari-
ability across species than RWC thresholds did (Table 3). On
average, the CV for thresholds based on Ψ
leaf
were four-fold
higher than those based on RWC.
Correlations among thresholds of water status at leaf
functional decline
Across species, most RWC thresholds for functional decline
tended to be intercorrelated (rvalues ranged from 0.64 to 0.99;
untransformed data; Table S8). In particular, the RWC inducing
50% declines of rehydration capacity (RWCPLRC50 ) was corre-
lated with other thresholds for hydraulic decline and dehydration
tolerance traits. Thus, RWCPLRC50 was correlated with the RWC
thresholds for declines of leaf hydraulic conductance by 10, 20,
50, and 80% (K
leaf 10
,K
leaf 20
,K
leaf 50
,K
leaf 80
, respectively;
Fig. 7a; Table S8), and the RWC thresholds for decline of outside
xylem hydraulic conductance K
ox
by 50% and 88% (Fig. 7b;
Table 4). None of the thresholds for K
x
decline were related to
those for rehydration capacity (Table 4). RWCPLRC50 was also
correlated with RWC
TLP
(Fig. 7c), and RWC
TLP
was also
correlated with other dehydration tolerance traits, including
RWCKleaf 10 and RWCgs20;50 (Table S8). RWC thresholds for loss
of F
v
/F
m
(RWC
PLCF
) were uncorrelated with those for leaf
hydraulic decline and TLP (Table 4). However, RWCPLCF50 was
correlated with RWC thresholds of stomatal conductance decline
(Table 4). Thresholds for functional decline in terms of Ψ
leaf
were
also intercorrelated; that is, WPLRC10 with Ψ
TLP
(r=0.93;
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(a) (b)
(c) (d)
(e) (f)
(g) (h)
(i) (j)
Fig. 2 Response of percentage loss of
maximum quantum yield of photosystem II
(F
v
/F
m
; noted in the text as PLCF) over
decreasing relative water content (RWC) for
the 10 species studied. Gray points are
measured leaves and solid regression lines are
best-fit models. The dashed lines in each plot
indicate the RWC associated with 10%
(blue) and 50% (red) losses of F
v
/F
m
.***,
P≤0.001. (a–j) refer to different species in
the panel.
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P=0.003), Ψ
leaf
at K
leaf 50,80
and K
x 50,88
(Table S9), and Ψ
TLP
with several Ψ
leaf
thresholds for dehydration responses, including
those of PLRC, K
leaf
,K
ox
, and K
x
(Table S9).
Discussion
By analyzing measurements of dehydration impacts on meso-
phyll, hydraulic, and stomatal functions of a set of diverse
angiosperm species, our study provides a thorough sequence of
water status thresholds driving leaf functional decline (Fig. 8).
We show for the first time that leaf damage as shown by loss of
rehydration and light-harvesting capacities occur with dehydra-
tion beyond severe failure of hydraulic function. Further, we
show an association of rehydration capacity decline with drought
sensitivity traits such as TLP and leaf hydraulic vulnerability.
Species showed much stronger variability and diversification in
Ψ
leaf
thresholds than RWC did.
Leaf rehydration capacity, a potential indicator of drought
tolerance
We found strong decline in rehydration capacity during dehydra-
tion. The decline of rehydration capacity with RWC was best
described by a sigmoid function for most species, suggesting that
leaf rehydration capacity is unaffected at mild dehydration levels.
A first stable phase was followed by an abrupt decrease in rehy-
dration capacity, which would result from irreversible loss of cell
volume or the death of cells. The second stable phase, which
occurs at very low RWCs, may represent apoplastic water intake
by capillarity, which would be independent of the dysfunction of
the parenchyma mesophyll tissue. Rehydration surveys in excised
and intact Laurus nobilis plants using X-ray microtomography
found water refilling of the xylem and apoplast by capillary forces
in excised samples, but seldom in intact plants (Knipfer et al.,
2017). An avenue for future research is the development of meth-
ods to quantify leaf PLRC under in vivo conditions to verify the
rehydration dynamics shown by rehydration curves and the
mechanisms involved in the loss of rehydration capacity.
Thresholds of F
v
/F
m
decline and its lack of post-dehydration
recovery
Water content decline had a strong impact on the maximum
quantum yield of fluorescence. Most species showed an exponen-
tial decrease of F
v
/F
m
during dehydration, with a rapid drop c.
20% RWC, suggesting significant damage late in dehydration
stress. This finding is consistent with a previous study on
Arabidopsis, which showed declines of F
v
/F
m
at a very similar
RWC threshold (Woo et al., 2008). Several previous studies have
suggested that leaf photochemistry is relatively stable under water
stress (Shabala & Pang, 2007; Flexas et al., 2009), as declines in
PSII photochemistry occur at lower water contents than g
s
and
TLP (Boyer & Potter, 1973; Miyashita et al., 2005). Moreover,
significant declines in F
v
/F
m
occur after the complete loss of
midrib xylem hydraulic function in sunflower plants (Cardoso
et al., 2018). We importantly extended these findings showing
Fig. 3 Maximum quantum yield of the photosystem II (F
v
/F
m
)of10
angiosperm species at different stages of water stress. Ranges of leaf
relative water content (RWC) for each water status are included in
parentheses. Bars represent mean (SE) values at full turgor (white bars),
dehydrated (light gray), and after rehydration (dark gray). Letters indicate
significant differences at a 0.95 confidence level.
Fig. 4 Differences between indices of percentage loss of Chl fluorescence
(F
v
/F
m
) of dehydrating leaves measured under low (7 2lmol m
2
s
1
;
gray boxes) vs high (343 42 lmol m
2
s
1
; white boxes) irradiance.
Colored points represent index values for each species. Measurements
were carried out on a subset of four species: Cercocarpus betuloides (red),
Comarostaphylis diversifolia (green), Lantana camara (blue), and
Malosma laurina (purple). Boxes indicate median, quartiles, minimum and
maximum values for each index of Chl fluorescence decline. Dashed
horizontal lines represent the occurrence of turgor loss point (TLP), and the
50% losses of stomatal conductance g
s50
, leaf hydraulic conductance
K
leaf 50
, and rehydration capacity PLRC
50
averaged for the four measured
species. ns, nonsignificant differences between the mean values of each
index under different irradiances (paired t-test across species; P≤0.05).
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that photochemical damage is reached only after full hydraulic
disruption in a range of diverse species. The F
v
/F
m
declines occur
at extremely low RWC and Ψ
leaf
, demonstrating that the light-
harvesting function of the leaf is compromised at much lower
water status than those triggering substantial loss of hydraulic
function. Indeed, PSII maximum quantum yield was the last
function to decrease in the sequence of dehydration-induced
damage. Notably, desiccation-tolerant plants, also known as ‘res-
urrection’ plants, show similar rapid declines of F
v
/F
m
when
reaching RWC of c. 20% (Beckett et al., 2012; Zia et al., 2016),
suggesting a common dehydration threshold for major photo-
chemistry damage even for species with photosynthetic repair
capabilities.
Our experiments on the 10 species focused on F
v
/F
m
in the
ambient light of the laboratory specifically to isolate the effect of
dehydration on leaf photochemistry without excess light and
temperature stresses. We note that high irradiance can induce
faster photoinhibition, ultimately leading to greater photochemi-
cal damage (Bj€orkman, 1987). Indeed, our measurements on a
subset of four species showed an empirical, nonsignificant ten-
dency for photochemistry dysfunction to occur earlier when
leaves were dehydrated under high irradiance. However, even
under high irradiance, F
v
/F
m
declines are known to occur at very
strong levels of water stress (Flexas et al., 2009). Our experiments
confirmed that photochemical decline in leaves dehydrated under
low or high irradiance occurs after major hydraulic dysfunction.
Fig. 5 Leaf functional impairment over a
gradient of decreasing total relative water
content. Red bars are mean values for each
threshold. Boxes show the 25
th
and 75
th
percentiles, and bars indicate extreme values.
Colors illustrate different thresholds of leaf
conductance (K
leaf
,K
x
,K
ox
; blue), stomatal
conductance (g
s
; yellow), turgor loss point
(TLP; orange), loss of rehydration capacity
(PLRC; green), and loss of F
v
/F
m
(PLCF;
purple). Numbers next to each threshold are
percentages of function loss. K
leaf
,K
ox
,K
x
,
and TLP values were obtained from Scoffoni
et al. (2011, 2017), Guyot et al. (2012), and
Bartlett et al. (2012b). Values for each
measured threshold are provided in Table 2
and Supporting Information Table S10.
Pairwise independent t-test comparisons
between all of the thresholds included in this
sequence are available in Table S6.
Fig. 6 Leaf functional impairment over a
gradient of decreasing water potential. Red
bars are mean values for each threshold.
Boxes show the 25
th
and 75
th
percentiles,
and bars indicate minimum and maximum
values. Colors illustrate different thresholds
of leaf conductance (K
leaf
,K
x
,K
ox
; blue),
stomatal conductance (g
s
; yellow), turgor
loss point (TLP; orange), and loss of
rehydration capacity (PLRC; green).
Numbers next to each threshold are
percentages of function loss. K
leaf
,K
ox
,K
x
,
and TLP values were obtained from Scoffoni
et al. (2011, 2017), Guyot et al. (2012), and
Bartlett et al. (2012b). Values for each
threshold are provided in Supporting
Information Table S11. Pairwise independent
t-test comparisons between all of the
thresholds included in this sequence are
available in Table S7.
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Further, when K
leaf
and g
s
declines are considered under low
irradiance, they show lower maximum values, and lower sensitiv-
ity to dehydration (Guyot et al., 2012). Thus, the sequence of
impacts of leaf dehydration is expected to be maintained
under different light irradiances. We observed no recovery of
F
v
/F
m
after rehydration, suggesting irreversible impairment of
photochemical activity as previously shown in crops undergoing
various levels of water stress (Giardi et al., 1996; Souza et al.,
2004). Dehydration-induced photochemical decline, and its lack
of recovery, may result from injuries to thylakoid membranes or
proteins (Hincha et al., 1987; Tambussi et al., 2000; Hoekstra
et al., 2001; Sharkey, 2005). We tested whether the decrease in
F
v
/F
m
might be associated with Chl degradation associated with
dark-induced senescence (H€ortensteiner, 2006), as previously
reported for excised barley leaves maintained well hydrated in
darkness (Scheumann et al., 1999). However, our measurements
showed that total Chl concentration was maintained in our
experimental dehydration treatment. In addition to its effect on
photochemistry, water scarcity primarily affects photosynthesis
by reducing CO
2
diffusion to the chloroplast due to declines of
stomatal and mesophyll conductance (Flexas et al., 2008), with
possible additional impacts on carbon reaction biochemistry
(Tezara et al., 1999; Grassi & Magnani, 2005). Future work is
needed to clarify the impacts of dehydration on the entire photo-
synthetic system in relation to the leaf stomatal, hydraulic, wilt-
ing, and damage thresholds included in this study.
Correlations among thresholds of dehydration-induced leaf
functional decline
We observed significant intercorrelation of most drought-
tolerance thresholds across species. This finding supports the
hypothesis for the coordination among thresholds proposed in a
meta-analysis of a smaller set of responses (Bartlett et al., 2016),
and extends this coordination to many more leaf drought-
tolerance traits. Such correlations among the thresholds for func-
tional declines with dehydration would represent co-selection for
drought tolerance, or mechanistic trait linkages. For instance, we
observed correlations of RWCPLRC50 with all the RWC thresholds
of leaf hydraulic conductance (RWCKleaf ), and outside-xylem
hydraulic conductance (RWCKox ). The relationship of thresholds
for declines in K
ox
and rehydration capacity is consistent with the
extra-xylary pathways representing a locus for the control of water
relations during dehydration, influenced by aquaporins that
Table 3 Ranges and variation of drought tolerance thresholds.
Index
RWC
CV (%)
RWC range
min/max (%) ΨCV (%)
Ψrange
min/max (MPa)
SLRT
statistic
(P-value)
K
leaf 20
2.9 92/99 78.1 1.34/0.08 232***
g
s20
6.2 85/>99 88.8 1.88/0.01 228***
K
ox 50
6.4 83/98 92.5 2.80/0.09 225***
g
s50
7.6 83/>99 83.6 2.72/0.04 230***
K
leaf 50
6.5 82/98 78.5 2.85/0.26 232***
g
s80
12.1 73/98 80.9 3.93/0.33 225***
TLP 3.9 78/89 30.3 3.45/1.18 224***
K
leaf 80
12.9 63/94 67.8 5.25/0.59 222***
K
ox 88
16.9 57/95 75.2 6.20/0.36 229***
K
x50
8.5 70/89 61.5 5.60/0.87 226***
PLRC
10
14.3 57/84 32.4 3.82/1.63 229***
K
x88
12.3 53/80 53.7 8.40/1.60 221***
PLCF
10
53.4 25/99 na na na
PLRC
50
9.9 34/45 na na na
PLCF
50
52.4 5/29 na na na
Coefficients of variation (CVs) and range of values of relative water
content (RWC) and water potential (Ψ
leaf
) thresholds of functional decline.
Thresholds are ordered from highest to least sensitive in terms of RWC
mean values. Note that high sensitivity in some thresholds was found due
to estimation of Ψ
leaf
thresholds using maximum likelihood statistical
functions and extrapolating high maximum values for stomatal
conductance and hydraulic conductance for fully hydrated leaves
(Ψ
leaf
=0 MPa), and RWC thresholds were estimated from Ψ
leaf
thresholds
using pressure–volume curves (see the Materials and Methods section).
See Table 1 for a list of abbreviations and units. na, not available, lack of
Ψ
leaf
thresholds. SLRT, signed-likelihood ratio test for equality of CVs.
***,P≤0.001.
(a) (b) (c)
Fig. 7 Relationships of the relative water content (RWC) inducing 50% loss of rehydration capacity with RWC thresholds of decline in leaf hydraulic
conductance (a), outside-xylem hydraulic conductance (b), and leaf turgor loss point (TLP) (c) across species. Values of decline in outside-xylem hydraulic
conductance were available only for eight of the species studied. K
leaf
,K
ox
and TLP values were obtained from Scoffoni et al. (2011, 2017), Guyot et al.
(2012), and Bartlett et al. (2012b). Standardized major axes regressions are included. *,P≤0.05; **,P≤0.01.
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would modulate the flow of water during transpiration and also
during cell rehydration (Scoffoni et al., 2017). Further, we
observed a correlation of RWCPLRC50 and WPLRC10 with RWC
TLP
and Ψ
TLP
, respectively. These correlations may arise from the
impact of turgor loss in the mesophyll and more specifically on
the outside-xylem hydraulic pathway (Scoffoni et al., 2014),
which would
constrain leaf rehydration capacity. We also observed a relation-
ship across species between leaf hydraulic declines and WPLRC10 .
This relationship is consistent with previous studies that empha-
sized the importance of leaf hydraulic failure in limiting func-
tional recovery after rewatering (Brodribb & Cochard, 2009;
Skelton et al., 2017b). However, this correlation may also be
driven by a trait co-selection exerted by water stress on both traits
for dehydration tolerance and resilience. We found a strong asso-
ciation of the RWC inducing a 50% decline of F
v
/F
m
, with the
thresholds of g
s
decline across the species studied, indicating that
different tissues are integrated in their tolerance of dehydration
(chloroplasts in the mesophyll and guard cells in the epidermis).
Thresholds for functional decline during dehydration in leaf
relative water content vs leaf water potential
All the measured dehydration tolerance thresholds showed less
variability in terms of RWC than of Ψ
leaf
. A previous study
reported narrower variability in stomatal conductance responses
to RWC than in Ψ
leaf
for soybean (Glycine max) and maize (Zea
mays) (Bennett et al., 1987). A narrow variability in RWC thresh-
olds may represent convergence in the hydration levels necessary
to protect mesophyll cells, and ultimately the chloroplasts, from
loss of function and damage. Despite the advantages of Ψ
leaf
for
the plant physiologist (e.g. the typically higher precision of Ψ
leaf
measurement, and its direct representation of the driving forces
for water movement), RWC can be a valuable water status indica-
tor of cell stress, as it represents relative cell volume shrinkage,
and thus tension on the cell and vacuolar membranes, cytoskele-
ton and cell wall and cell solute concentrations (Sack et al.,
2018). Our study illustrates how different water status indices
should be considered in plant–water relations studies and espe-
cially assessed for their value as proxies for dehydration-induced
responses.
The sequence of dehydration-induced functional decline
We provided an empirically based sequence of functional and
structural damage in response to leaf water stress. Our sequence
of Ψ
leaf
thresholds is similar for the three traits in common with
those considered in the meta-analysis of Bartlett et al. (2016);
that is, g
s50
,K
leaf 50
, and TLP occur sequentially as leaves dehy-
drate. Notably, the water status thresholds for 50% declines in
g
s
and K
leaf
were not significantly different, consistent with
coordination on average between leaf hydraulic decline (which
depends strongly on the outside-xylem pathways) and stomatal
closure (Brodribb & Holbrook, 2003; Scoffoni & Sack, 2017).
During leaf dehydration, g
s50
occurred significantly before the
declines in leaf xylem conductance K
x50
, consistent with recent
reports of stomatal closure preceding the appearance of
embolism in grapevines (Hochberg et al., 2017). On average
across species, RWCgs80 occurred at similar water status as
RWC
TLP
(87% 3.31 vs 86% 1.05, respectively; Table S6),
consistent with mild cell dehydration as a driver of ABA accu-
mulation and stomatal closure (McAdam & Brodribb, 2016;
Sack et al., 2018). The sequential occurrence of g
s80
and TLP
suggests that significant stomatal closure may act to prevent
mesophyll damage.
On average across species, PLRC
10
occurred at lower RWC
and Ψ
leaf
than TLP did, and at similar water status to K
x88
,
which represents major vein xylem embolism (Scoffoni et al.,
2016) and which, for stem xylem, has been proposed as a
hydraulic threshold indicating irreversible drought damage (Urli
et al., 2013). Notably, PLRC
10
, along with the majority of the
hydraulic traits included in the sequence, occurred at thresholds
less negative than the 4 MPa maximal boundary of absolute
stomatal closure (Martin-StPaul et al., 2017). PLRC
50
occurred
at significantly lower water status, demonstrating that partial leaf
rehydration capacity exists even beyond significant mesophyll
damage and hydraulic dysfunction.
The sequence established in this study of 10 diverse
angiosperm species indicates an ‘outside to inside’ progression of
overall leaf sensitivity to dehydration through the leaf tissues and
cells. Stomatal closure and outside-xylem hydraulic decline tend
to begin with mild dehydration and to peak at around TLP, with
xylem embolism occurring later, followed by substantial
Table 4 Correlations of the relative water content (RWC) indices of loss of rehydration capacity and Chl fluorescence (F
v
/F
m
) with other RWC indices of
leaf functional impairment.
RWC
TLP
RWCK10 RWCK20 RWCK50 RWCK80 RWCKox50 RWCKox88 RWCKx50 RWCKx88 RWCgs20 RWCgs50 RWCgs80
RWCPLRC10 0.53 0.59 0.62 0.57 0.48 0.72*0.59 0 0.09 0.51 0.58 0.55
RWCPLRC20 0.57 0.64*0.66*0.61 0.52 0.72*0.6 0.02 0.15 0.5 0.58 0.58
RWCPLRC50 0.65*0.80** 0.80** 0.78** 0.75*0.79*0.84** 0.1 0.26 0.33 0.43 0.54
RWCPLCF10 0.37 0.19 0.10 0.07 0.03 0.01 0.03 0.22 0.26 0.31 0.3 0.3
RWCPLCF20 0.44 0.27 0.18 0.12 0.01 0.07 0.01 0.37 0.2 0.46 0.46 0.42
RWCPLCF50 0.51 0.46 0.41 0.28 0.13 0.27 0.11 0.69 0.06 0.77** 0.8** 0.64*
Pearson’s correlation coefficients of bivariate cross-correlations. Bold values indicate significant correlations. n=10 for all variables, except K
ox
and K
x
indices, for which n=8. See Table 1 for a list of abbreviations and units. See Supporting Information Tables S8 and S9 for a full correlation matrix amongst
RWC and Ψ
leaf
thresholds of functional decline.
*,P≤0.05; **,P≤0.01.
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Phytologist Research 145

(a)
(b)
(c)
(d)
Fig. 8 Thresholds of dysfunction at different stages of leaf dehydration. (a) Fully operational leaf with a continual water flow through the leaf vein xylem
conduits, bundle sheath, and spongy parenchyma (blue dashed line). (b) A 50% decrease of outside-xylem and stomatal conductance. (c) Occurrence of
vein xylem embolism inducing a 50% decrease of xylem-specific conductance, and loss of turgor inducing significant changes in leaf thickness and damage
to mesophyll cells. At this stage, there is a strong reduction of the stomatal aperture, severely diminishing stomatal conductance. TLP, turgor loss point.
(d) Severe dehydration inducing disruption of the hydraulic function, major damage in mesophyll cells, loss of rehydration capacity, and a decreased
photochemical activity in the chloroplast. PLCF, percentage loss of Chl fluorescence. Thresholds occurrences are marked by red letters. Circled numbers
indicate the order of occurrence of each dehydration-induced impact on leaf function. Symbols and definitions of thresholds are provided in Table 1.
Relative water content (RWC) and Ψ
leaf
thresholds of functional loss and analyses of sequence occurrences are provided in the Supporting Information.
New Phytologist (2019) 223: 134–149 Ó2019 The Authors
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146

irrecoverable damage, as indicated by loss of rehydration capac-
ity, and, finally, irreversible damage to the photochemical system
in the chloroplast (Fig. 8). Species with greater overall drought
tolerance show shifts of multiple elements of the system to better
resist declines during dehydration. Notably, by the time that sub-
stantial damage has occurred to the leaves, many functions have
declined, and so estimating the early impacts of drought on water
balance or photosynthesis should focus on hydraulic traits rather
than photodamage. A distinction between ‘dehydration’ and ‘des-
iccation’ has been proposed to occur at a RWC of 30–40% based
on the initiation of major physiological and molecular changes
(Zhang & Bartels, 2018), and most extant angiosperms cannot
survive the dehydration of their vegetative tissues to 20–30%
RWC (Oliver et al., 2010). Our findings show that major physio-
logical processes decline in the dehydration stage, as for the 10
study species we observed a major decrease of hydraulic functions
before 40% RWC, and only F
v
/F
m
function persisted below the
40% boundary. Given that declines in F
v
/F
m
seem to occur at the
very end of the dehydration-induced injury sequence, our study
suggests that the functions of the PSII are maintained beyond the
point at which photosynthetic CO
2
assimilation and hydraulic
functioning are impaired.
Our findings provide a baseline sequence for the declines of
functions to be confirmed in larger comparisons of plant species
and functional types. Further, these observations should be con-
sidered in studies relying on decline in chloroplast function as an
indicator of drought effects at the leaf scale when ChlF is used as
an indicator of drought impact, since declines in ChlF occur at
the late stages of water stress (Yao et al., 2018). Based on our
findings, such declines of leaf photochemistry performance would
correspond to irreversible damage to leaf functionality, beyond
stomatal closure, leaf vein embolism, and irreversible loss of rehy-
dration capacity.
Acknowledgements
We thank the staff of the Mildred E. Mathias Botanical Garden
of UCLA for providing access to the living collection and grant-
ing permission to collect samples, and Alec Baird, Marvin
Browne, Leila Fletcher, Christian Henry, and Victor Lu for
thoughtful comments on an earlier version of the paper. We
thank Nate McDowell and four anonymous referees for their
insightful comments. This work was supported by a UC
MEXUS-CONACYT postdoctoral research fellowship to ST and
the National Science Foundation (grant 1457279).
Author contributions
ST and LS designed the research. RP performed measurements
of stomatal conductance. CS contributed data of leaf hydraulic
vulnerability. SDD and GPJ contributed to the design of mea-
surement protocols. ST performed experiments, analyzed the
data, and wrote the first draft of the manuscript with contribu-
tions from LS. All authors contributed to subsequent revisions of
the manuscript.
ORCID
Grace P. John https://orcid.org/0000-0002-8045-5982
Lawren Sack https://orcid.org/0000-0002-7009-7202
Christine Scoffoni https://orcid.org/0000-0002-2680-3608
Santiago Trueba https://orcid.org/0000-0001-8218-957X
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Supporting Information
Additional Supporting Information may be found online in the
Supporting Information section at the end of the article.
Fig. S1 Total chlorophyll concentration during leaf dehydration
based on SPAD measurements.
Fig. S2 Leaf functional impairment over a gradient of decreasing
total relative water content (RWC) for the ten measured species
Table S1 Pressure-volume curve parameters for 10 analyzed
angiosperm species.
Table S2 Dataset containing all variables measured for 10 ana-
lyzed angiosperm species.
Table S3 Model results for the determination of leaf relative
water content (RWC) inducing percentage loss of rehydration
capacity (PLRC).
Table S4 Model results for the determination of total relative
water content (RWC) inducing percentage loss of maximum
quantum yield of PSII (PLCF) under different irradiance.
Table S5 Results of the linear mixed-effect model analysis for
chlorophyll fluorescence responses during leaf dehydration and
rehydration.
Table S6 Independent t-tests for differences in RWC thresholds
of leaf drought tolerance traits.
Table S7 Independent t-tests for differences in Ψ
leaf
thresholds of
leaf drought tolerance traits.
Table S8 Correlations between RWC indices of leaf functional
impairment.
Table S9. Correlations between Ψ
leaf
indices of leaf functional
impairment.
Table S10 RWC thresholds of drought tolerance traits.
Table S11 Ψ
leaf
thresholds of drought tolerance traits.
Please note: Wiley Blackwell are not responsible for the content
or functionality of any Supporting Information supplied by the
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.
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