Research

Drought response strategies define the relative contributions

of hydraulic dysfunction and carbohydrate depletion during tree
mortality
Patrick J. Mitchell1, Anthony P. O’Grady1, David T. Tissue2, Donald A. White3, Maria L. Ottenschlaeger1 and
Elizabeth A. Pinkard1
1
CSIRO Ecosystem Sciences, Climate Adaptation Flagship, Private Bag 12, Hobart, Tas., 7001, Australia; 2Hawkesbury Institute for the Environment, University of Western Sydney, Bourke
Street, Richmond, NSW, 2753, Australia; 3CSIRO Ecosystem Sciences, Climate Adaptation Flagship, Private Bag 5, Wembley, WA, 6913, Australia

Summary
Author for correspondence:
Plant survival during drought requires adequate hydration in living tissues and carbohydrate
Patrick J. Mitchell reserves for maintenance and recovery. We hypothesized that tree growth and hydraulic
Tel: +61 3 6237 5624
strategy determines the intensity and duration of the ‘physiological drought’, thereby affect-
Email: patrick.mitchell@csiro.au
ing the relative contributions of loss of hydraulic function and carbohydrate depletion during
Received: 2 August 2012 mortality.
Accepted: 20 October 2012

We compared patterns in growth rate, water relations, gas exchange and carbohydrate
dynamics in three tree species subjected to prolonged drought.
New Phytologist (2013) 197: 862–872
Two Eucalyptus species (E. globulus, E. smithii) exhibited high growth rates and water-use
doi: 10.1111/nph.12064 resulting in rapid declines in water status and hydraulic conductance. In contrast, conservative
growth and water relations in Pinus radiata resulted in longer periods of negative carbon
Key words: carbon limitation, drought balance and significant depletion of stored carbohydrates in all organs. The ongoing demand
mortality, hydraulic conductance, hydraulic for carbohydrates from sustained respiration highlighted the role that duration of drought
failure, nonstructural carbohydrates, plant plays in facilitating carbohydrate consumption.
strategy.
Two drought strategies were revealed, differentiated by plant regulation of water status:
plants maximized gas exchange, but were exposed to low water potentials and rapid hydraulic
dysfunction; and tight regulation of gas exchange at the cost of carbohydrate depletion.
These findings provide evidence for a relationship between hydraulic regulation of water sta-
tus and carbohydrate depletion during terminal drought.

capacity to regulate its carbon and water balance under drought

Introduction
Changes in temperature and rainfall patterns across many forest
and woodland ecosystems are thought to underlie the increasing
vulnerability of tree species to drought-related mortality (Allen
et al., 2010). Global analyses of forest vulnerability and drought-
related mortality events across different biomes suggest that a
wide range of forest types are susceptible to periods of extreme
temperature and water deficit (Allen et al., 2010; Choat et al.,
2012). However, in many cases, mortality rates vary among spe-
cies or functional types (Pook et al., 1966; Breshears et al., 2005;
Fensham & Fairfax, 2007) suggesting strong differences in
drought resistance among co-occurring species. Despite major
progress on understanding on how water deficit affects plant
functioning (Sperry, 2000; Breda et al., 2006; Flexas et al., 2006)
the controls on species survival under extreme drought remain
poorly resolved and limit our ability to adequately predict future
changes in ecosystem structure and function.
Recent syntheses have built on earlier work on drought physi-
ology to describe how mechanisms of mortality are linked to plant
conditions of differing intensity and duration (McDowell et al.,
2008; Allen et al., 2010). These frameworks suggest that, for any
given drought event, both plant hydraulic strategy and meteoro-
logical conditions will define the ‘physiological drought’ experi-
enced by the plant. Physiological drought is a function of plant
regulation of water-use in response to declining soil water
potential and thresholds associated with hydraulic- or carbohy-
drate-mediated mortality. For example, plants exposed to low-
intensity but long-duration droughts may maintain water status
above critical water potential thresholds but deplete stored carbo-
hydrates to lethal limits (i.e. carbon starvation). Conversely,
under high-intensity drought, incapacity to regulate plant water
status above critical thresholds will promote xylem cavitation and
death through dehydration (i.e. hydraulic failure). In addition to
these drivers, trees often sustain tissue damage from biotic and
abiotic agents such as pest outbreaks (Ayres & Lombardero,
2000) and extreme temperatures (De Boeck et al., 2010) that can
amplify the impacts of drought. This conceptual framework has
been the focus of intense debate within the literature (Adams

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et al., 2009; McDowell & Sevanto, 2010; Sala et al., 2010) but
has not been extensively tested with observational and experi-
mental data.
The processes of hydraulic failure and carbon starvation are
intimately linked (McDowell et al., 2011) via the balancing act
required to minimize water loss and maximize carbon uptake at
the leaf surface. Adjustments in stomatal aperture act to reduce
transpiration in response to declining hydraulic conductance
and/or reductions in leaf turgor and help plants avoid water
potentials that can induce hydraulic failure (Sperry, 2000). At the
whole-plant scale, the hydraulic architecture of plants appears to
be well coordinated with photosynthetic capacity (Brodribb &
Feild, 2000; Santiago et al., 2004; Quentin et al., 2012)., Under
favourable growing conditions, higher photosynthetic capacity
tends to promote higher growth rates (Poorter et al., 1990;
Lambers & Poorter, 1992). Therefore, higher rates of hydraulic
conductance may promote high growth rates in trees by main-
taining high stomatal conductance (gs), internal [CO2] and car-
bon gain (Tyree, 2003). However, during protracted periods of
water deficit, high hydraulic conductance may also increase plant
susceptibility to cavitation (Maherali et al., 2006). Evidence for a
growth vs hydraulic safety trade-off is highlighted by relation-
ships between wood density and growth rate (Enquist et al.,
1999; Muller-Landau, 2004) and cavitation safety (Hacke et al.,
2001). The capacity for rapid growth (i.e. high intrinsic relative
growth rates) can reduce carbon allocation to hydraulic safety
(e.g. reduced lumen fraction, thicker cell walls), potentially making
these species more vulnerable to hydraulic failure during
drought.
Maintenance of cell turgor, which is a hydraulically mediated
process, plays an important role in regulating the carbon balance
of plants. Growth is particularly sensitive to changes in cell turgor
and often declines before reductions in leaf photosynthesis in
response to drought (Hsiao et al., 1976; Amthor & McCree,
1990) In contrast, respiration tends to be relatively insensitive to
drought and does not decline proportionally in response to
declining photosynthesis (Atkin & Macherel, 2009; Ayub et al.,
2011). As a result, the concentration of nonstructural carbohy-
drates within plant tissues depends on the balance between car-
bon supply (i.e. photosynthesis) and carbon demand (i.e. growth
and respiration). In the short term, the concentration of non-
structural carbohydrates during drought will increase if reduc-
tions in growth precede declines in photosynthesis, a response
that has been observed in many species (Tissue & Wright, 1995;
K€orner, 2003; Ayub et al., 2011). However, if drought is
prolonged, reductions in carbon assimilation and subsequent con-
sumption of reserve carbohydrates may occur until a threshold
is reached, after which plants may die of carbon starvation
(McDowell et al., 2011).
As a result of these trade-offs in hydraulic and carbon dynam-
ics, plants operate along a continuum of responses to drought,
which are characterized by varying levels of hydraulic regulation
to declining water availability associated with structural and
physiological traits (Mitchell et al., 2008; Bartlett et al., 2012).
We propose that physiological drought will vary in species with
different growth and hydraulic strategies and generate water stress
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conditions of differing duration and intensity that are associated
with mortality processes involving hydraulic failure and carbo-
hydrate starvation. To test this, we compared the magnitude and
timing of changes in water relations, gas exchange and carbohy-
drate dynamics in three tree species with different water use and
growth characteristics exposed to a terminal drought treatment.
We hypothesized that tight hydraulic regulation of water and car-
bon uptake reduces the risk of rapid hydraulic failure, but results
in increased duration of the physiological drought and promotes
depletion of nonstructural carbohydrates.
Materials and Methods
Plant material and growth conditions
Whole-plant responses to drought were studied using three spe-
cies with different patterns in growth and drought response.
Eucalyptus globulus (Labill.) is a fast-growing tree with profligate
water use. It is an important native and plantation forest species
in south-eastern Australia, and planted extensively for wood and
pulp products in temperate and Mediterranean climates around
the globe. Anecdotal evidence suggests that Eucalyptus smithii
(R.T. Baker), which has similar wood properties to E. globulus, is
less susceptible to drought than E. globulus and as a result is being
trialled as a replacement for this species in some regions. Pinus
radiata (D. Don) is the most widely planted pine species in the
world and an important softwood plantation species. Overall,
growth and water-use in P. radiata is more conservative than in
eucalypt species, based on stand-level comparisons of growth and
water-use (Myers et al., 1996).
One hundred seedlings of each of the three species were
obtained for this experiment. Eucalyptus globulus seedlings were
obtained from the Forestry Tasmania Nursery near Perth,
Tasmania. Seedlings of E. smithii were obtained from seed sown
and grown at the Western Australia Plantation Resources nursery,
Manjimup, Western Australia. Pinus radiata cuttings were
sourced from Lanoma Estate, Westerway, Tasmania. The euca-
lypt seedlings and P. radiata cuttings were c. 6 months and
18 months old, respectively, at the start of the drought treatment
and observations of root systems during harvest suggest that all
species had explored the majority of the soil volume. Seedlings/
cuttings were planted into a potting mix consisting of eight parts
composted pine bark to three parts coarse river sand. The euca-
lypt mix contained low phosphorus premium controlled-release
fertilizer (N : P : K – 17.9 : 0.8 : 7.3) and P. radiata were fertilized
with a standard controlled-release fertilizer (N : P : K – 19.4 :
1.6 : 5). Three hundred 250-mm diameter black pots (8 l) were
numbered and filled with the potting mix to a combined weight
(pot plus mix) of 6 kg and wetted to field capacity. The mix
content of each pot was compressed to 6 l volume. One eucalypt
seedling or pine cutting was planted into each numbered pot.
Plants were grown in two adjacent glasshouses where growth
temperatures were allowed to vary as a function of outside tem-
perature and ranged from 3 to 38°C, with a mean of 18.1°C.
Half of the seedlings from each of the three species were allocated
randomly to each glasshouse. The position of seedlings within
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both glasshouses was changed every 2 wk. There were no signifi-
cant differences between glasshouses in any of the physiological
parameters among the three species (see later); thus, the data were
pooled for subsequent analyses.
Watering regime
Equal numbers of plants from each species were randomly allo-
cated to one of two treatments (control/well-watered or drought).
Plants in the control treatment were maintained at field capacity
throughout the experiment utilizing daily drip irrigation. All con-
trol plants were watered for 30 min daily at a rate of 4 l h1. The
drought treatment was designed to achieve a gradual dry-down of
the soil by applying a watering regime that replaced 80–90% of
the water lost via evapotranspiration during the previous 24 h.
Every 2 wk, six randomly selected plants from each species
were assigned to a separate bench and used as representative sam-
ples for calculating the watering regime. On a daily basis, these
plants were weighed ( 0.1 g; Sartorius BP1600S, Goettingen,
Germany) between 16:00 h and 18:00 h. We estimated the total
amount of water lost via evapotranspiration (including evapora-
tion from bare soil) as the difference between the previous day’s
pot mass (after watering) minus the current day’s pot mass. To
smooth out day-to-day variation in this water loss, the actual vol-
ume of water replaced on a daily basis was calculated as a running
mean of the previous 3 d. Water supply to the plants was gradu-
ally reduced until < 10 ml of water was being added to each plant
on a daily basis. The drought-treated plants received water using
this method for c. 60, 80 and 120 d for E. globulus, E. smithii and
P. radiata, respectively. After this point, no water was added and
pots were allowed to dry until symptoms of plant death were
noted. In our study, plants were considered to be dead when all
leaf material had turned brown, stems were brown and respira-
tion rates in the stem were zero (as estimated by gas exchange
measurements described below). To confirm our diagnosis, a sub-
set of plants exhibiting these symptoms was rewatered and did
not recover after > 1 month of rewatering.
Gas exchange
Leaf gas exchange measurements were conducted on mature, fully
expanded leaves from six plants per treatment and species using a
portable gas exchange system (Licor-6400; Licor, Lincoln, NE,
USA). Light-saturated photosynthesis (Asat) was measured with a
standard leaf chamber equipped with blue–red light-emitting
diodes at a photosynthetic photon flux of 2000 lmol m2 s1
and [CO2] of 390 ll l1. Leaf-to-air vapour pressure deficit dur-
ing measurements varied between 1 kPa and 1.5 kPa, and leaf
temperature was maintained at c. 20°C. The Asat measurements
were conducted mid-morning (09:00–12:00 h) at 2–4 wk inter-
vals throughout the course of the experiment.
Dark respiration rates (Rdark) were measured at 20°C on
detached leaves (sampled immediately before measurement) from
six plants per treatment that had been placed in a dark tempera-
ture-controlled room for at least 2 h before measurement. The
Rdark measurements were conducted on the same day as Asat using
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a large transparent conifer chamber attached to the same gas
exchange system (Licor 6400-05). Stem respiration or lack of (used
to assess tree death) was measured on dark-adapted stems using the
same chamber and gas exchange system. Stems were clamped using
the middle of the conifer chamber and the gasket stem interface
was sealed using Blu Tack (Bostik International, Paris, France).
Soil-to-leaf hydraulic conductance
Soil to leaf hydraulic conductance was estimated at 2–4 wk inter-
vals to provide an estimate of the extent to which whole-plant
water transport was affected by reductions in hydraulic conduc-
tance via cavitation in xylem tissues. Total soil to leaf hydraulic
conductance (KP, mmol m2 s1 MPa1) was estimated as:
E
KP ¼ Eqn 1
ðWsoil  Wleaf Þ
where E is whole-plant transpiration (mmol m2 s1) and bulk
soil water potential, Ψsoil (MPa), and leaf water potential, Ψleaf
(MPa), are estimated from predawn and midday leaf water
potential, respectively (Whitehead & Jarvis, 1981).
On the evening before the day of measurements, six plants
from each species in each treatment were randomly selected. Each
pot was wrapped in plastic bags and sealed around the base of the
stem to prevent soil evaporation during the following day. Over-
night, the above-ground portion of each plant was totally covered
in large plastic bags to ensure that there was no nocturnal transpi-
ration and to bring plant water potential into equilibrium with
soil water potential.
Approx. 1 h before sunrise, the plastic bags covering above-
ground biomass were removed. Pre-dawn leaf water potential
(Ψpd) of one leaf from each plant was measured using a Scholand-
er-type pressure chamber (PMS instruments, Corvalis, OR, USA).
Transpiration (E) was estimated by weighing each of the measure-
ment pots at midday. After the pots were weighed, a leaf from each
plant was sampled for the determination of midday Ψleaf. After c.
1 h, each of the pots were reweighed. Transpiration was calculated
as the weight lost over the measurement interval (making sure to
account for the weight of the leaf material removed for determina-
tion of Ψleaf); thus, E could be expressed as mmol (H2O) m2(leaf
area) s1. The height and diameter of each plant was recorded and
leaf area estimated from allometric relationships developed from
destructive sampling of the plants (see later for details).
Pressure-volume analysis
Leaf pressure-volume curves were generated on leaves from six
control plants per species using the bench drying technique
(Turner, 1988). Briefly, leaves were sampled between 07:00 h
and 09:00 h from well-watered plants and rehydrated for 3–4 h
in the dark. Before commencing measurements, petioles were
recut under water and blotted dry. Timing of measurements of
leaf weight ( 0.1 mg) and Ψleaf was done to ensure adequate
points during the initial phase of rapid water loss and 3–5 points
on the linear phase (post-turgor loss) of the curve. The leaf water
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potential at turgor loss (ΨTLP) and relative water content at tur-
gor loss for each sample was calculated using a pressure–volume
curve-fitting routine (v 5.5; K. Tu, University of California
Berkeley, CA, USA) based on the template of Schulte & Hinckley
(1985).
Destructive harvesting
Six plants from each species and treatment combination were
destructively harvested at approximately monthly intervals from
beginning of the experiment until 2–4 wk after death. These
plants were used to determine relative growth rate (RGR) and
develop allometric relationships used to estimate leaf area for cal-
culating E (n = 36; r2 = 0.67–0.81) and subsampled for carbohy-
drate analyses. Harvested plant biomass was divided into leaves,
stem and roots. A sample of five to ten leaves was used for deter-
mination of specific leaf area (SLA). Leaf area was determined
using a scanner and software (WinRhizo; Reagent Instruments,
Quebec City, QC, Canada). Immediately after harvesting, each
biomass component was placed into an oven at 110°C for 1 h to
kill any biological activity. Plant material was then subsequently
dried to a constant weight at 65°C.
RGR (mg g1 d1) was determined using the ‘classical’
method (Hunt, 2002) whereby RGR was calculated across one
harvest interval using:
ð1=W ÞðdW =dt Þ ¼ ð1=La ÞðdW =dt Þ  La =Lw  Lw =W
RGR ULR SLA LWF
Eqn 2
Where ULR is the unit leaf rate; LWF is leaf weight fraction;
W is plant dry weight, La is leaf area; Lw is leaf dry weight. Rela-
tive growth rates were calculated using an excel worksheet
described in detail by Hunt (2002).
Carbohydrate analyses
Subsamples of oven-dried material from the destructive harvests
were ground to a fine powder in a ball mill. Soluble carbohydrates
were extracted from c. 20 mg of dried plant tissue in 5 ml of 80%
aqueous ethanol (v : v) in a polyethylene tube. The mixture was
boiled in a water bath at 95°C for 30 min, and then centrifuged
at 3000 rpm for 5 min. The supernatant was collected and the
pellet re-extracted once with 5 ml of 80% aqueous ethanol (v : v)
and once with 5 ml of distilled water then boiled and centrifuged
as before. The supernatants were reserved and evaporated (until
Table 1 Summary of differences in leaf turgor loss point (ΨTLP) and corresponding relative water content (brackets), relative growth rate (RGR) and soil-
to-leaf hydraulic conductance (KP) during predrought conditions and day of drought at mortality for each species ( 1 SE)
Species ΨTLP (MPa) RGR (mg g1 d1)
Eucalyptus globulus 2.22 (0.77)  0.04a 0.024  0.006a
Eucalyptus smithii 2.00 (0.88)  0.05b 0.028  0.012a
Pinus radiata 1.66 (0.87)  0.09c 0.011  0.008b
Superscript letters denote groupings of species based on pairwise comparisons (P < 0.05).
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1–3 ml remained) in a rotational vacuum concentrator at 40°C.
Total soluble sugars were determined on the supernatants follow-
ing the anthrone method (Ebell, 1969). Total starch was deter-
mined on the remaining pellets after the ethanol and water
extractions and assayed enzymatically using a total starch assay kit
(Megazyme International Ireland Ltd, Wicklow, Ireland). Soluble
sugar (SS) and starch (St) concentrations (mg g1) were calcu-
lated as content of the measured pool divided by dry weight of
the sample. Whole-plant SS and St was calculated as the sum of
the weighted concentrations (concentration multiplied by the
proportion of component dry mass to total dry mass) of the dif-
ferent biomass components (leaf, stem and root). Total nonstruc-
tural carbohydrate (TNC) concentrations were calculated as the
sum of whole plant SS and St.
Statistical analysis
One-way analyses of variance (ANOVA) were performed to test
for between-species differences in predrought RGR, KP and
ΨTLP (P < 0.05). Differences between treatments in Ψpd and gas
exchange parameters (Asat and Rdark) for each species were tested
using a two-way ANOVA using day of drought and treatment as
variables (P < 0.05). A two-way ANOVA was used to test for
treatment differences in TNC, SS and St among plant organs
(leaf, stem, and root) at mortality for each species. Changes in
TNC, SS and St concentrations within different treatments
between the first biomass harvest (predrought conditions) and
the final harvest (2–4 wk after all plants were dead) were calcu-
lated (% change in concentration at mortality vs concentration
before drought minus 100) to determine accumulation vs deple-
tion of carbohydrates in each biomass component. Between-
treatment differences were then examined using a two-way ANOVA
(P < 0.05) using organ and treatment as factors. Student’s t-tests
were performed on whole-plant TNC at mortality to test for
treatment differences (P < 0.05). All analyses were performed in
R (v2.13.0; Foundation for Statistical Computing).
Results
Plant mortality
The mean time-to-mortality for each species, based on the
complete desiccation of above-ground biomass (and associated
symptoms), ranged from c. 90 d for E. globulus to c. 215 d for
P. radiata (Table 1). Time-to-mortality among individuals within
the same species varied from 1–2 wk for the eucalypt spp. to
2–3 wk for P. radiata.
KP (mmol m s1 MPa1) Day of drought at mortality
4.43  0.14a 90
5.76  0.14b 130
2.31  0.18c 215
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Growth rates and plant water relations
Mean total dry biomass and leaf area before the initiation of the
drought was 67 g and 0.17 m2 for P. radiata, 34 g and 0.20 m2
for E. globulus, and 20 g and 0.13 m2 for E. smithii, respectively.
The two eucalypt species had similar RGR during predrought con-
ditions (0.024–0.028 mg g1 d1; Table 1). In contrast, P. radiata
had significantly lower RGR (P < 0.05; 0.011 mg g1 d1;
Table 1). The ΨTLP showed a contrasting pattern and was highest
in P. radiata (1.6 MPa) and lowest in E. globulus (2.2 MPa),
while E. smithii was intermediate (2.0 MPa; P < 0.05, Table 1).
Relative water content at turgor loss was also lowest in E. globulus
(0.77; Table 1) and higher in the other two species (0.87; Table 1).
We used the time taken for Ψpd to reach ΨTLP to gauge how
quickly water deficit progressed in these three species, based on
the average progression of leaf water status using fitted linear
functions of day of drought vs natural logarithm (Loge) (*1)
Ψpd (r2 = 0.80–0.89). Despite E. globulus having a lower ΨTLP,
the time taken for Ψpd to reach this threshold ΨTLP was the
shortest (44 d) of the three species. Eucalyptus smithii had a
slightly higher ΨTLP, but it took 68 d for Ψpd to reach this ΨTLP.
Pinus radiata had the highest ΨTLP, but spent 93 d above the
ΨTLP (Fig. 1a). The decline in Ψpd post-turgor loss was very
rapid in the eucalypt species. Between-tree variability in Ψpd was
low until Ψpd were between 4.0 MPa and 4.5 MPa for the
eucalypt species; below these values, within- and between-plant
(a) (b)
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variation became very large, which was indicative of loss of
hydraulic conductance across much of the canopy. All species
reduced whole-plant E (as % of max E in control plants) to c. 5%
at the ΨTLP while Kp (expressed as a% of max Kp in control
plants) was reduced by < c. 20%.
Before the onset of drought, KP was higher in the two eucalypt
species (4.4–5.8 mmol s1 m2 MPa1) than in P. radiata
(2.3  0.2 mmol m2 s1 MPa1; Table 1). While KP declined in
the well-watered eucalypt control plants throughout the course of
the experiment (Fig. 1b), the decline in KP in response to the
drought treatment was more marked and declined linearly with
days of drought. This rate of decline was more rapid in E. globulus
and E. smithii (0.05 mmol m2 s1 MPa1 d1 and 0.04
mmol m2 s1 MPa1 d1, respectively) than in P. radiata; in con-
trast to the eucalypt species, the rate of decline in KP with the pro-
gression of the drought in the pine was best described by a negative
exponential function (P < 0.01, Fig. 1b). Based on these relation-
ships and the Ψpd data, we estimated that KP became zero
at c. 3.5 MPa for the eucalypt spp. and reached very low values
(< 0.05 mmol m2 s1 MPa1) in P. radiata at c. 2.4 MPa.
P. radiata displayed comparatively tighter hydraulic regulation,
whereby KP (expressed as a percentage of control KP) declined more
rapidly with changes in midday leaf water potential (Ψmd) than the
eucalypt species (Fig. 2). These patterns were also reflected in the
rate of water loss in response to declining water availability, as indi-
cated by the slope of the predawn Ψleaf vs Loge E, which was largest
Fig. 1 Progression of plant water relations for
control (closed symbols) and droughted (open
symbols) Eucalyptus globulus, Eucalyptus
smithii and Pinus radiata seedlings. (a) Day of
drought and mean predawn leaf water
potential (Ψpd,  1 SE). (b) Day of drought
and soil-to-leaf hydraulic conductance (KP,
 1 SE). The start of the drought treatment is
denoted by the vertical dotted line. The KP
data are fitted with a linear (eucalypt spp.) or
exponential decay (P. radiata, P < 0.05) for
E. globulus (drought; y = 0.05x + 3.11,
r2 = 0.88 and control; ns), E. smithii (drought;
y = 0.04x + 3.27, r2 = 0.92 and control
y = 0.04x + 5.04, r2 = 0.51) and P. radiata
(drought; y = 2.16 9 e0.02x, r2 = 0.88 and
control; y = 4.00 9 e0.01x, r2 = 0.38). The leaf
water potential at turgor loss (ΨTLP) for each
species is shown in the left panels (horizontal
dash-dot line).
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Fig. 2 Relationship between mean midday leaf water potential (Ψleaf) and
soil-to-leaf hydraulic conductance (KP), expressed as a percentage of
control for each sampling occasion. The leaf water potential at turgor loss
(ΨTLP) for each species (Eucalyptus globulus, Eucalyptus smithii and Pinus
radiata) is shown as a vertical dashed line. Data are fitted with a three-
parameter sigmoidal function; y = a/(1 + e((xc)/b)) (P < 0.05).
for P. radiata (2.1 mmol m2 s1 MPa1) and significantly smaller
(P < 0.05) for the eucalypts (1.0 mmol m2 s1 MPa1 and
0.8 mmol m2 s1 MPa1 for E. globulus and E. smithii, respec-
tively; Fig. 3). These data suggest that regulation of water loss in
P. radiata was more sensitive to changes in soil water availability
than in the two eucalypts.
Gas exchange
Predrought, Asat was highest in E. smithii (Asat = 16.1 
0.5 lmol m2 s1), intermediate in E. globulus (10.5  0.6 lmol
m2 s1) and lowest in P. radiata (3.5  0.3 lmol m2 s1;
Fig. 4). These patterns were also reflected in Rdark for the three
species. Eucalyptus smithii had the highest respiration rates
(1.8  0.13 lmol m2 s1), E. globulus had intermediate rates
(0.5  0.03 lmol m2 s1) and the lowest rates were in
P. radiata (0.2  0.01 lmol m2 s1; Fig. 4). Both Asat and Rdark
of the well-watered control plants declined during the course of
the experiment, reflecting natural reductions in these parameters
with shorter days and onset of winter. Nonetheless, drought sig-
nificantly reduced Asat relative to the control treatment in all spe-
cies; E. globulus reached zero (as estimated given the accuracy and
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Fig. 3 Relationship between mean predawn leaf water potential (Ψpd) and
whole-plant transpiration (E, natural log transformed) for the three study
species. Each species data set are fitted with a linear regression (P < 0.01);
Eucalyptus globulus (red circles); y = 1.03x + 1.32, r2 = 0.88, Eucalyptus
smithii (green squares); y = 0.76x + 1.06, r2 = 0.86 and Pinus radiata (blue
diamonds); y = 2.12x + 1.18, r2 = 0.90.
precision of the gas analyser) at c. 50 d, followed by E. smithii (c.
80 d) and then P. radiata (c. 100 d, Fig. 4). The number of days
of drought until plants reached a point of zero carbon assimila-
tion was similar to the time taken to reach ΨTLP among all three
species (see previous section). In contrast, for the majority of the
drought (up until c. 2 wk before mortality), Rdark was not signifi-
cantly different between treatments for any of the species
(P < 0.05). These patterns in gas exchange resulted in increased
Rdark: Asat during the drought sequence in all three species (data
not shown). We used the day of drought when Asat of each
species reached zero as an estimate of the date when plants experi-
enced a negative carbon balance; that is, we used this date to
assume that carbon assimilation was less than whole-plant carbon
utilization (Fig. 4). We found that E. globulus died c. 40 d after
Asat c. 0, followed by E. smithii (c. 50 d) and P. radiata (c. 115 d).
This duration of negative carbon balance was similar to the
period of time droughted plants remained at zero turgor among
the three species (i.e. Ψleaf < ΨTLP).
Changes in nonstructural carbohydrate concentrations
The depletion or accumulation of St and SS was represented as
the proportional change in carbohydrate concentration at the
time of mortality relative to carbohydrate concentration before
the initiation of the drought treatment. For the control (well-
watered) plants, there was a consistent pattern of accumulation of
St in the roots of all species over the duration of the experiment.
There was also significant accumulation of St in the stem and
leaves in control P. radiata plants (Fig. 5, P < 0.05) and in the
leaves of E. smithii. In contrast, the drought treatment promoted
significant depletion of leaf St in all species at mortality, although
for the two eucalypt species this depletion was largely offset by
significant accumulation of SS (Fig. 5, P < 0.05). Pinus radiata
showed significant depletion in the root and stem St pools and
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(a) (b)

Fig. 4 Progression of leaf carbon assimilation

of control (closed symbols) and drought
(open symbols) seedlings of Eucalyptus
globulus, Eucalyptus smithii and Pinus
radiata. For each species, changes in mean
light-saturated leaf photosynthesis (Asat,
 1 SE) is shown in the left panel and mean
leaf dark respiration (Rdark,  1 SE) is shown
in the right panel. The vertical lines (dash–
dot) denote estimated points along the
drought sequence that net leaf carbon
assimilation became zero or negative.

(a) (b)

Fig. 5 Percentage change in starch and

soluble sugar concentrations in root, stem
and leaf components in drought (red) and
control (blue) seedlings of the three study
species (Eucalyptus globulus, Eucalyptus
smithii and Pinus radiata). Changes in
carbohydrate concentrations for each
component are calculated by dividing
concentration at mortality by concentration
before the start of the drought treatment
(100%). Positive values denote accumu-
lation and negative values represent
depletion of carbohydrates during the
experiment. Asterisks denote significant
differences in carbohydrate concentrations
between initial and final harvests using
P < 0.05.

leaf and stem SS pools relative to initial conditions (Fig. 5) and a all biomass pools in droughted P. radiata plants relative to con-
significant depletion in whole-plant TNC concentration in the trol plants (Table 2). However, there was no significant depletion
P. radiata plants at mortality (48%; Fig. 6). Furthermore, in St or SS in the roots or stems of either eucalypt species relative
TNC concentrations were, on average, 41% lower at mortality in to pre-drought conditions. These temporal patterns in

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(a) than depletion owing to drought (Fig. 5, Table 2). Furthermore,

there were no significant changes in the whole-plant TNC con-
centrations at mortality in the eucalypt species (Table 2).

Relationships between water status and carbohydrates

Regulation of plant water status modifies the duration and inten-
sity of the physiological drought and subsequent consequences
for whole-plant carbon balance at mortality (Fig. 6). Based on
both water relations and patterns in carbon assimilation and utili-
zation under drought, the following relationships were observed.
Eucalyptus globulus experienced a short but intense drought, as
(b) measured by the time taken for Ψpd to fall below the ΨTLP. In
contrast, P. radiata experienced a longer, less intense drought;
E. smithii was intermediate between these extremes (Fig. 6).
Whole-plant carbohydrate status was strongly linked to these dif-
ferences in duration of drought , where both eucalypt species
showed no significant depletion in TNC at the whole-plant level,
whereas P. radiata showed markedly less TNC at mortality com-
pared with the well-watered control (Fig. 6).

Fig. 6 Conceptual diagram of the relationship between water status and
carbohydrate depletion for the three study species (Eucalyptus globulus;
red lines/circles, Eucalyptus smithii; green lines/squares and Pinus radiata;
blue lines/diamonds) during drought. (a) The trajectory of predawn leaf
water potential (Ψpd) in response to drought (based on fitted regression
equations of the three species) and (b) percentage depletion in total
nonstructural carbohydrates (TNC, weighted by components) in drought
plants at mortality (relative to predrought values). Arrows extending from
the three species symbols on the Ψleaf trajectories to the average estimate
of mortality (vertical dashed line) represent the time over which different
species remained in a state of neutral or negative carbon balance before
mortality.
carbohydrate concentrations indicate that the lower root St and
SS in the droughted eucalypt plants at mortality were mostly a
result of accumulation of these pools in the control plants rather
Discussion
Species responses to drought are primarily determined by the rate
and degree to which plant water status is hydraulically regulated.
Subsequently, this behaviour mediates the duration and intensity
of the physiological stress and the processes that underpin mortal-
ity. Eucalypt species were characterized by high growth rates and
water use, which intensified physiological drought through a
rapid depletion of soil water and resulted in plants reaching a
water status that induced complete loss of hydraulic function. In
contrast, the conservative growth and water-use strategies of
P. radiata reduced water loss early in the drought and reduced
assimilation rate but led to significant depletion of whole plant
TNC and prolonged the time to tree death. These responses rep-
resent very different strategies along the drought response contin-
uum and support predictions of McDowell et al. (2008),
whereby the drought conditions and associated physiological
adaptations can alter the relative contributions from hydraulic
dysfunction and the exhaustion of available carbohydrates during
mortality.

Table 2 Carbohydrate concentration of starch (St) and soluble sugars (SS, mg g1 DW  1 SE) for different organs in each species treatment at the final
harvest

Leaf Stem Root Whole plant Whole plant

St SS St SS St SS St SS TNC

Eucalyptus globulus Control 27.6 (6.7) 68.1 (4.2) 3.8 (1.0) 28.5 (2.8) 7.9 (1.5) 38.2 (4.3) 14.8 (2.0) 45.6 (1.9) 60.4 (2.6)
Drought 4.3 (0.8) 123.0 (5.8) 2.1 (0.3) 26.6 (3.8) 2.8 (0.4) 30.6 (3.5) 2.9 (0.2) 62.5 (4.3) 65.4 (4.5)

Eucalyptus smithii Control 55.3 (22.2) 70.6 (5.0) 2.6 (0.3) 26.7 (4.5) 13.5 (3.2) 44.6 (6.7) 20.1 (5.0) 41.7 (2.9) 61.9 (7.6)
Drought 3.7 (0.7) 103.3 (19.3) 2.8 (0.6) 28.3 (2.1) 3.0 (0.4) 27.4 (2.2) 2.9 (0.3) 51.0 (5.1) 53.9 (4.9)
Pinus radiata Control 22.4 (10.0) 54.8 (3.4) 11.6 (6.7) 33.1 (3.3) 31.0 (12.2) 35.1 (2.2) 19.0 (5.0) 35.3 (1.7) 54.3 (6.2)
Drought 5.4 (1.1) 33.2 (1.9) 1.5 (0.9) 21.0 (3.0) 2.2 (0.5) 47.4 (4.5) 2.8 (0.4) 30.8 (0.8) 33.7 (0.8)

Whole plant St, SS and total non-structural carbohydrates (TNC) represent a weighted average for plant organ (based on the contribution of each organ to
total biomass). Bold text denotes significant differences between control and drought treatments (P < 0.05).

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Hydraulic responses to drought: the higher they fly, the
harder they fall
Lower ΨTLP in the eucalypt species enabled carbon assimilation
to continue to lower leaf water potentials than droughted
P. radiata (Table 1, Fig. 4). Hydraulic regulation of plant water
status appeared to be well coordinated with ΨTLP, with all species
showing similar reductions in Kp at the ΨTLP (Fig. 2). This
pattern is consistent with observed responses of stomatal conduc-
tance and leaf hydraulic conductance during desiccation
(Brodribb & Holbrook, 2003). In many cases, lower ΨTLP indi-
cates greater tolerance to drought (Bartlett et al., 2012), presumably
because species may extract soil water at lower water potentials,
while avoiding stomatal closure. However, under sustained
drought, keeping stomata open to lower water potentials, as was
the case in the two eucalypt spp., exposes species to increased risk
of hydraulic failure. Accordingly, Ψpd in the two eucalypt species
declined quickly following turgor loss in response to the deple-
tion of soil water, thereby resulting in xylem cavitation and rela-
tively rapid mortality (90–130 d). The Ψpd value at which KP
became zero was estimated to be c. 3.5 MPa and accords well
with field studies of E. globulus that found Kp reached zero at c.
3.7 MPa, with death occurring soon after, thereby demonstrating
strong evidence for hydraulic failure at these water potentials
(D. A. White et al., unpublished data). This was also supported
by the dramatic increase in within-canopy and between-plant
variation in leaf water potential at similar water potentials (Fig. 1)
suggesting a breakdown in hydraulic connections within the can-
opy. In contrast, P. radiata exhibited rapid declines in KP at
higher ΨTLP than the eucalypts (Fig. 3), thereby reducing gas
exchange and the rate of decline in Ψpd. This more conservative
drought response strategy in P. radiata resulted in more gradual
reductions in Kp, which approached very low values (2% of con-
trol) at higher values of Ψpd (2.4 MPa) after c. 130 d, yet
remained alive for a further c. 90 d despite the absence of any
watering. A greater sensitivity of stomata to reductions Kp in this
species may act to hydraulically isolate the vascular system from
the drying soil and slow the progression towards hydraulic failure
and dehydration.
Tighter regulation of water-use increased carbohydrate
depletion
In this experiment, strategies to regulate water-use affected both
the supply of carbon through stomatal closure and reduced car-
bon assimilation, while simultaneously lowering the demand for
carbon through reduced growth rates. The temporal sequence of
leaf gas exchange, where Asat is a proxy for rate of carbon uptake
and Rdark is a proxy for carbon usage, were used to characterize
the response of leaf gas exchange to drought and relate these tem-
poral dynamics to whole-plant carbon balance (Atkin et al.,
2007). The point at which drought reduced leaf carbon assimila-
tion to zero varied among species, although we acknowledge that
negative carbon balance may have been reached before this point.
However, Rdark was relatively insensitive to the drought in all
three species, except for a period of 1–2 wk before mortality,
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during which respiration declined significantly. These responses
are consistent with previous studies of respiratory responses to
drought that show that mitochondrial respiration is often main-
tained under drought (Atkin & Macherel, 2009). The majority
of these previous studies, however, were short-duration events of
between 5 and 30 d (Atkin & Macherel, 2009). Under our
extended drought (90–215 d), Rdark was generally unchanged
even when leaf carbon balance was negative for substantial peri-
ods. Consequently, this ongoing respiratory demand for carbohy-
drates in leaves was reflected in significant depletion of leaf St
among all species. These patterns in St depletion under drought
supports findings that identify starch pools as important integra-
tors of carbon supply and demand (Sulpice et al., 2009). The
eucalypt species had a concomitant accumulation in leaf SS that
could be attributed to the initial lag between reductions in
growth and carbon assimilation (Vandoorne et al., 2012) and the
maintenance of this pool for ongoing metabolic function.
The two eucalypt species and P. radiata exhibited different
patterns of carbohydrate utilization during extended drought.
Under well-watered conditions, the two fast-growing eucalypt
species accumulated starch in roots, but not in leaves or stems.
Starch accumulation did not occur under drought but whole-
plant starch reserves in the eucalypts were not apparently
depleted at mortality. In contrast, significant starch accumula-
tion was observed under well-watered conditions in P. radiata
but significant starch depletion was observed under drought in
all plant organs. The level of starch depletion in P. radiata was
very large, ranging from 54% to 60% among different plant
organs and 48% for the whole plant; at mortality, starch was c.
85% lower in drought plants compared with well-watered con-
trol plants. While it is very difficult to determine whether starch
depletion was sufficient to generate mortality, studies of respro-
uting plant species, where background concentrations of carbo-
hydrates are generally high (Pate et al., 1990), showed lethal
levels of starch depletion between 81% (Cruz et al., 2003) and
96% (Canadell & Lopez-Soria, 1998) compared with control
plants. However, starch depletion sufficient to induce mortality
may be less for non-resprouting species such as Pinus, which
typically do not accumulate large carbohydrate reserves. Indeed,
Galiano et al. (2011) reported extremely low levels of non-
structural carbohydrate concentrations in stem sapwood of dead
Scots pine trees (< 20% of living trees) after an extreme drought
event. Our study shows for the first time that prolonged periods
of negative carbon balance during drought causes significant
depletion in whole-plant TNC and provides evidence for carbo-
hydrate starvation as a contributing process for mortality, as pos-
tulated by McDowell et al. (2008). We have shown that for
significant exhaustion of carbohydrates to occur, plants must
remain below or near the point of negative carbon balance while
avoiding irreversible cavitation. These conditions are more likely
during low-intensity/long duration water deficit and/or in spe-
cies that exhibit more conservative hydraulic regulation of water
status.
Future research is needed to elucidate whether duration of
drought alone determines gross depletion of carbohydrates at
mortality, or if carbohydrate utilization and mobilization are
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impeded at a threshold water status (Sala et al., 2010), suggesting
an interaction between drought intensity and duration. A broader
ecological question is the extent to which growth rate and related
traits define the size of carbon reserves and patterns in utilization
and mobilization, and therefore the vulnerability of different spe-
cies. If inter-specific differences in species source-to-sink strength
ratios are related to RGR, this may provide a basis for predicting
how plant carbon balance is affected under drought or other
stressors. Our study highlights the importance of understanding
how whole-plant regulation of water status determines the expo-
sure period and degree to which a favourable carbon balance may
be compromised, which may increase plant vulnerability to addi-
tional biotic and abiotic factors.
Interplay between mortality processes and drought
response strategy
Our results demonstrate that the positioning of a species along
the drought response continuum are characterized to a large
degree by growth and maximum photosynthetic rates, hydraulic
conductance and turgor loss point. These traits influence the
level of hydraulic regulation of water status (intensity) and time
to mortality (duration) – the two key parameters likely to under-
lie the degree of hydraulic dysfunction and carbohydrate deple-
tion during severe drought stress (Fig. 6). The ‘wait it out’
strategy of P. radiata resulted in a longer duration to mortality,
but also significant depletion of carbohydrate reserves. This strat-
egy may lead to death during drought or significantly reduce the
capacity to recover from sublethal drought. Under long-duration
physiological drought, trees become increasingly vulnerable to
losing carbohydrate sources (i.e. leaves and branches) via leaf
shedding (Galiano et al., 2011) or biotic agents such as insect
defoliation (Carnicer et al., 2011). In contrast, in the eucalypt
species the short duration to mortality resulted in depletion of
the carbohydrate reserves in the leaves only; no depletion in stor-
age tissues such as roots and stems was observed. However,
where drought conditions induce rapid and significant losses of
hydraulic conductance, but not tree death (Kursar et al., 2009),
recovery processes involving growth of new leaves and water-
conducting tissues (Brodribb et al., 2010) or potentially the
refilling of embolized conduits (Bucci et al., 2003) may require
significant utilization of stored carbohydrates. Where successive
drought cycles of this kind persist, tree survival in the long term
may ultimately depend on the availability of stored carbohy-
drate.
Differences in physiological mechanisms and the associated
ecological strategies among species have broad implications for
ecosystem change and may be viewed in terms of their resistance
and resilience to drought and related disturbances (Westman,
1978; MacGillivray et al., 1995). In ecological terms, the ‘wait
it out’ strategy of P. radiata is characteristic of resistance strate-
gies, where survival is reliant on strong regulation of water sta-
tus associated with conservative growth patterns and structural
and physiological traits. However, this strategy offers little
capacity for recovery once critical carbohydrate thresholds have
been exceeded. Alternatively, the eucalypt species typifies a
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resilience-type strategy, where rapid loss of hydraulic function
means that they succumb to drought rapidly. However, under
non-lethal conditions (e.g. low intensity and/or short duration
events), plants have the potential to recover by building new
hydraulic pathways and leaves through utilization of remaining
carbohydrate reserves. The differential impacts of drought on
species and ecosystem dynamics may be better understood by
elucidating the role of ecological strategy in underpinning plant
responses to droughts of differing intensity, duration and fre-
quency.
Acknowledgements
We acknowledge the assistance of Dale Worledge, David Page
and Dominik Laschinger with biomass harvesting and Renee
Smith and Kaushal Tewari with carbohydrate analyses. Funding
was provided by the Australian Government’s Department of
Agriculture, Forestry and Fisheries. Michael Battaglia, Jenny
Carter and Victor Resco de Dios provided useful feedback and
comments on earlier versions of this manuscript. We also thank
the reviewers for their helpful comments and feedback.
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New Phytologist Ó 2012 New Phytologist Trust