Journal of Experimental Botany, Vol. 60, No. 8, pp. 2379–2390, 2009
doi:10.1093/jxb/erp071 Advance Access publication 25 March, 2009
RESEARCH PAPER
The role of mesophyll conductance during water stress and
recovery in tobacco (Nicotiana sylvestris): acclimation or
limitation?
Alexander Galle*, Igor Florez-Sarasa, Magdalena Tomas, Alicia Pou, Hipolito Medrano, Miquel Ribas-Carbo
and Jaume Flexas
Grup de Recerca en Biologia de les Plantes en Condicions Mediterrànies, Departament de Biologia (UIB-IMEDEA), Universitat de les Illes
Balears, Carretera de Valldemossa km 7.5, 07122 Palma de Mallorca, Spain
Received 24 November 2008; Revised 17 February 2009; Accepted 19 February 2009
Abstract
While the responses of photosynthesis to water stress have been widely studied, acclimation to sustained water
stress and recovery after re-watering is poorly understood. In particular, the factors limiting photosynthesis under
these conditions, and their possible interactions with other environmental conditions, are unknown. To assess these
issues, changes of photosynthetic CO2 assimilation (AN) and its underlying limitations were followed during
prolonged water stress and subsequent re-watering in tobacco (Nicotiana sylvestris) plants growing under three
different climatic conditions: outdoors in summer, outdoors in spring, and indoors in a growth chamber. In
particular, the regulation of stomatal conductance (gs), mesophyll conductance to CO2 (gm), leaf photochemistry
(chlorophyll fluorescence), and biochemistry (Vc,max) were assessed. Leaf gas exchange and chlorophyll fluorescence data revealed that water stress induced a similar degree of stomatal closure and decreased AN under all three
conditions, while Vc,max was unaffected. However, the behaviour of gm differed depending on the climatic conditions.
In outdoor plants, gm strongly declined with water stress, but it recovered rapidly (1–2 d) after re-watering in spring
while it remained low many days after re-watering in summer. In indoor plants, gm initially declined with water stress,
but then recovered to control values during the acclimation period. These differences were reflected in different
velocities of recovery of AN after re-watering, being the slowest in outdoor summer plants and the fastest in indoor
plants. It is suggested that these differences among the experiments are related to the prevailing climatic
conditions, i.e. to the fact that stress factors other than water stress have been superimposed (e.g. excessive light
and elevated temperature). In conclusion, besides gs, gm contributes greatly to the limitation of photosynthesis
during water stress and during recovery from water stress, but its role is strongly dependent on the impact of
additional environmental factors.
Key words: Drought, mesophyll conductance, Nicotiana sylvestris, photosynthetic limitation, recovery, stomatal conductance,
water stress.
Introduction
Limited water availability adversely affects plant productivity, growth, and survival (Boyer, 1982; Chaves et al., 2003;
Flexas et al., 2006a), in particular with regard to the
expected higher frequency of drought events due to rapid
changes in climate (Luterbacher et al., 2004; Schär et al.,
2004). Thus, strategies of tolerance, adaption, and survival
* To whom correspondence should be addressed. E-mail: [email protected]
Abbreviations: AN, maximum net CO2 assimilation rate; Ci, substomatal CO2 concentration; Cc, chloroplast CO2 concentration; gs, stomatal conductance for CO2;
gm, mesophyll conductance for CO2; C*, CO2 compensation point under non-photorespiratory conditions; J, photosynthetic electron transport rate; PPFD,
photosynthetic photon flux density; Rd, leaf respiration in the dark; RWC, relative leaf water content; Ta, air temperature; Tl, leaf temperature; Vc,max, maximum
velocity of carboxylation.
ª The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
2380 | Galle et al.
will be of major importance for plants growing under
adverse environmental conditions. Numerous studies have
clearly shown that water shortage or water stress primarily
affects photosynthetic CO2 assimilation and therefore sets
a limit to plant productivity and growth (Quick et al., 1992;
Flexas et al., 2002; Lawlor and Cornic, 2002; Monclus
et al., 2006; Galle et al., 2007; Haldimann et al., 2008).
Water stress-induced stomatal closure has been shown to
act as the initial and most prominent limitation to CO2
assimilation, as diffusion of CO2 from the atmosphere to
the sites of carboxylation in the chloroplast is impaired.
However, the impairment of photosynthetic CO2 assimilation during water stress may not be exclusively explained by
stomatal resistance to CO2 diffusion, as limitations may
also results from leaf internal resistances. These consist of
the CO2 pathway from the intercellular airspaces to the
mesophyll cells, the chloroplasts, and the sites of carboxylation (Flexas et al., 2008). Although the contribution of
each of the leaf internal resistances to the total limitation of
CO2 assimilation is still unclear, several methods have been
designed to assess the overall internal resistance or internal
conductance for CO2 diffusion, also called mesophyll
conductance (gm) (Evans et al., 1986; Harley et al., 1992;
Ethier and Livingston, 2004; Warren, 2006; Sharkey et al.,
2007; Flexas et al., 2008; Warren, 2008).
Many studies have shown that gm is finite and greatly
variable among different species (Flexas et al., 2008).
Moreover, gm may change in response to climatic variables;
for example, gm decreased under water stress (Flexas et al.,
2002; Grassi and Magnani, 2005; Flexas et al., 2006b;
Galmes et al., 2007b), salt stress (Delfine et al., 1999;
Centritto et al., 2003), low nitrogen availability (Warren,
2004), and varying CO2 concentrations (Flexas et al.,
2007b). These findings support the idea of an important
role for gm in the photosynthetic response of plants to
climatic constraints.
Additionally, metabolic impairments and/or cell damage
may also affect photosynthesis, especially under severe
water stress (Mittler, 2002; Reddy et al., 2004) and,
particularly under conditions favouring oxidative stress,
such as when water stress is combined with high light and
temperature (Flexas et al., 2006b; Zhou et al., 2007). The
contribution of these stomatal and non-stomatal processes
to the adaptation to water stress can vary among species, as
well as with the intensity and duration of imposed stress.
Furthermore, the recovery phase after relief of stress (i.e.
rainfall or irrigation) becomes another important part of the
overall plant physiological response to a water stress period.
The capability for photosynthetic recovery from an extreme
water stress event determines future growth and survival of
plants in their habitat. Notably, to date, little is known
about the underlying processes of photosynthetic recovery
from water stress; however, very recently this topic has
gained more and more attention (Ennahli and Earl, 2005;
Miyashita et al., 2005; Flexas et al., 2006a; Galle et al.,
2007; Galmes et al., 2007b). As this is of substantial
importance for plants in general and within the context of
future climate change in particular, more studies are needed
to understand the physiological basis of recovery from
water stress. Moreover, processes that limit recovery of
photosynthesis seem to be key factors in understanding
what makes some plants withstand and survive drought
better than others. A way to assess photosynthetic limitation processes during water stress and recovery has been
proposed by Grassi and Magnani (2005), who divided the
total limitation into three components: limitation by
stomatal and mesophyll diffusion, as well as by biochemical
processes (i.e. carboxylation activity). From the results of
their quantitative limitation analysis on ash and oak trees in
the field they could explain, for example, that the high nonstomatal limitation during summer drought was mainly due
to restrictions of CO2 diffusion within the mesophyll. These
findings underline the importance of gm during stress
conditions and further suggest an important contribution
to the overall adaptation of plants to drought stress.
However, possible short-term changes of gm during water
stress and recovery were not followed, as gm was monitored
only on several days throughout three vegetation periods
(years) with different summer precipitation. Other shortterm water stress experiments have also shown a decrease
of gm (Ennahli and Earl, 2005; Galmes et al., 2007b),
indicating a general trend of decline under water stress and
remaining high resistance during re-watering. However, and
apart from a possible role for aquaporins (Terashima
and Ono, 2002; Uehlein et al., 2003; Flexas et al., 2006c) and
carbonic anhydrase (Badger and Price, 1994; Gillon and
Yakir, 2000) in facilitating CO2 diffusion at the cellular level,
little is known about the signals for gm regulation during
stress and in particular during adaptation and recovery
(Flexas et al., 2008).
In the present study, the main limiting factors of
photosynthesis during water stress and recovery were
analysed in tobacco (Nicotiana sylvestris L.), emphasizing
leaf internal diffusive and non-diffusive components in
order to improve the understanding of what facilitates plant
adaptation to drought. To assess the effect of additional
factors, such as climatic conditions (i.e. light and temperature), on water stress acclimation and recovery, experiments
on the effects of water stress and re-watering were carried
out on the same plant species under field conditions in
summer and spring, as well as under growth chamber
conditions.
Materials and methods
Plant growth conditions
Three experiments were carried out with 6- to 9-week-old
tobacco plants (Nicotiana sylvestris L.): (i) outside at the
experimental field of the University of the Balearic Islands
(Palma, Spain) during summer (June–August 2007) with
a maximum photosynthetic photon flux density (PPFD)
incident on leaves of >1900 lmol m2 s1 and maximum air
temperatures (Ta,max) reaching almost 36 C; (ii) outside
during spring (March–May 2008), with maximum PPFD of
Role of mesophyll conductance during water stress and recovery in tobacco | 2381
1600 lmol m2 s1, but most often being between 750 lmol
m2 s1 and 1250 lmol m2 s1, and Ta,max reaching 28 C;
and (iii) inside a growth chamber, with a constant PPFD of
600 lmol m2 s1 (12 h) and Ta,max of ;26 C. All plants
were grown in pots with a 3:1 mix of horticulture substrate
and perlite, and were well irrigated every day before the
start of each experiment (including supplementary nutrition
with Hoagland’s solution once a week). In all three cases,
water stress was imposed by withholding water until severe
water stress was reached. Severe water stress was considered
to have occurred when stomatal conductance for water
vapour dropped to <50 mmol m2 s1, according to
Medrano et al. (2002). Thereafter, plants were maintained
at this intensity of water stress for another week by adding
the amount of water they lost during the day. After this
period of stress acclimation, plants were consecutively rewatered to field capacity, and the recovery of photosynthetic traits was followed.
Plant water status
From the first day of withholding water until the first day of
re-watering, plants were weighed every day in the evening
(at ;19:00 h) and total loss of water was recorded. When
necessary, plants were irrigated with the amount of water
they had lost during the day (as indicated above).
At the beginning of the experiment, the first and the last
day of severe water stress, as well as during the first days of
re-watering, the relative water content (RWC) of 4–6 leaves
per treatment was calculated as:
RWCð%Þ¼1003ðFWDWÞ=ðTWDWÞ
where FW, TW, and DW denote the weight of fresh, turgid,
and dry leaf tissue, respectively. FW was determined
immediately after sampling, while TW was obtained after
incubating leaf discs in distilled water for 48 h in the dark at
4 C. DW was determined after 72 h in a drying oven at
;70 C.
Leaf gas exchange and chlorophyll a fluorescence
Throughout the experiments, maximum net CO2 assimilation (AN), gs, and chlorophyll a fluorescence were measured
simultaneously with an open infrared gas-exchange analyser
system (Li-6400; Li-Cor Inc., Lincoln, NE, USA) equipped
with a leaf chamber fluorometer (Li-6400-40; Li-Cor Inc.).
At least four measurements on the youngest fully expanded,
sun-exposed leaves of control and stressed plants were
carried out each day in the late morning (11:00–12:30 h)
under a light-saturating PPFD of 1500 lmol m2 s1
(provided by the light source of the Li-6400 with 10% blue
light). Recordings were taken at 30, 28, and 25 C in
summer-, spring-, and growth chamber-grown plants, respectively. The CO2 concentration in the Li-6400 leaf
chamber (Ca) was set to 400 lmol CO2 mol1 air, and the
relative humidity of the incoming air ranged between 40%
and 60%. CO2 response curves (‘AN–Ci curves’) were
performed for watered, non-watered, and re-watered plants
by varying the CO2 concentration around leaves that had
been previously acclimated to saturating light conditions
(;15–20 min at a PPFD of 1500 lmol m2 s1).
Due to the fact that tobacco plants grew fastest during
summer and hence ageing of leaves occurred more rapidly
particularly in control plants, leaves used for measurements
were changed twice during the experimental period to
provide young and full maturity leaves. Moreover, this
change was necessary to avoid self-shading of leaves.
From the fluorescence measurements, the actual quantum
efficiency of the photosystem II (PSII)-driven electron
transport (UPSII) was determined according to Genty et al.
(1989) as
UPSII ¼ ðFm #Fs Þ=Fm #;
where Fs is the steady-state fluorescence in the light (here
PPFD 1500 lmol m2 s1) and Fm# is the maximum
fluorescence obtained with a light-saturating pulse (;8000
lmol m2 s1). As UPSII represents the number of electrons
transferred per photon absorbed by PSII, the rate of
electron transport (J) can be calculated as
J lmol e m2 s1 ¼ UPSII PPFDa
where the term a includes the product of leaf absorptance
and the partitioning of absorbed quanta between PSI and
PSII. As shown in Fig. 1, a was determined for each
treatment from the slope of the relationship between UPSII
and UCO2 (i.e. the quantum efficiency of gross CO2
fixation), which was obtained by varying the light intensity under non-photorespiratory conditions in an
atmosphere containing <1% O2 (Valentini et al., 1995).
Due to the deviation from linearity in the UPSII and UCO2
relationship at low light intensities in spring (Fig. 1b),
only the linear part of the slope has been used for
determination of a. As the curvilinear part is in the range
of low light intensities, this may not affect the estimates at
the high light used during the measurements. The curvilinear shape has been already described by Genty et al.
(1989) and Seaton and Walker (1990), and was intensively
discussed by Öquist and Chow (1992). A faster decrease
of the photochemical yield of open reaction centres than of
Fv#/Fm# in low light intensities according to the model of
Butler (1978), some changes in alternative electron sinks
at different light intensities, and/or the differences in determining the parameters (chlorophyll a fluorescence is
recorded predominantly from chloroplasts close to the
upper part of the illuminated leaf, while UO2 is based on
O2 evolving from the whole leaf tissue) have been
considered the reason for this curvilinear nature. The a
values determined for control plants were 0.5, 0.46, and 0.47,
and for water-stressed plants were 0.5, 0.49, and 0.5 in
summer, spring, and the growth chamber experiments,
respectively.
The maximum quantum efficiency of PSII (Fv/Fm) at predawn was recorded frequently during the experiments as
described elsewhere (Maxwell and Johnson, 2000), using the
Li-6400 flurometer (Li-Cor Inc.).
2382 | Galle et al.
Fig. 1. Representative data of the relationship of the quantum yield of photosystem II (UPSII) and UCO2 [(AN+Rd)/PPFD]. Measurements
were carried out under low oxygen (<1%) and varying CO2 concentrations. These CO2 curves were performed in all three experiments on
leaves under severe drought (open symbols), and well-watered ‘control’ and re-watered conditions (filled symbols).
From combined gas-exchange and chlorophyll a fluorescence measurements, the mesophyll conductance for CO2
(gm) was estimated according to Harley et al. (1992) as
gm ¼ AN =fCi ½C ðJ þ 8ðAN þ Rd ÞÞ=½J 4ðAN þ Rd Þg;
where AN and Ci were obtained from gas-exchange
measurements. A value of 37.4 lmol mol1 for the CO2
compensation point under non-respiratory conditions (C*)
was used after Bernacchi et al. (2002) as determined for the
related species Nicotiana tabacum. Other Rubisco kinetics
and their temperature dependencies were also taken from
Bernacchi et al. (2002). Dark respiration (Rd) was determined with an isotope ratio mass spectrometer (IRMS)
at 25 C as described by Ribas-Carbo et al. (2005)
throughout the spring and growth chamber experiments. In
the summer experiment, Rd was determined by gasexchange measurements (n >4) at 28 C, after plants had
been dark-adapted for ;2 h during the afternoon. All
respiration data were corrected for temperature, using
a Q10 of 2.2 (see Materials and Methods in Valentini et al.,
1995).
Calculated values of gm were used to convert A–Ci curves
into A–Cc curves according to the following equation:
Cc ¼ Ci ðAN =gm Þ
Maximum velocity of carboxylation (Vc,max) was derived
from the A–Cc curves according to Bernacchi et al. (2002).
Corrections for leakage of CO2 into and out of the leaf
chamber of the Li-6400 were applied to all gas-exchange
data, as described by Flexas et al. (2007a). Due to low gs
values under severe water stress and a possibly increased
contribution of conductance via the cuticle (gc), estimates
of gs were corrected for gc as described elsewhere (Boyer
et al., 1997). In short, for each experimental condition, gas
exchange was measured across the leaf with its lower side
sealed with lubricant and an impermeable plastic foil to
hinder any gas exchange via its cuticle and stomata. The
obtained gc was multiplied by two to account for the upper
and lower side of the leaf, and Ci was recalculated based
on the new gs values (gs–gc), using the equations pro-
vided by the manufacturer (LI-6400 manual version 5,
Licor Inc.).
Quantitative limitation analysis
To assess the limitations imposed by water stress and
recovery on photosynthesis, a quantitative limitation analysis of photosynthesis was conducted for all three data sets
according to Grassi and Magnani (2005). According to their
approach, measurements of AN, gs, gm, and Vc,max or the
maximum capacity for electron transport (Jmax) were used
to calculate the proportion of the three major components
of total limitation for CO2 assimilation: stomatal (SL) and
mesophyll conductance (ML), as well as biochemical processes (BL). From previous AN–Cc curves, it was confirmed
that at ambient CO2 concentration, net photosynthesis was
always limited by Vc,max and not Jmax, regardless of the
treatment. Therefore, Vc,max was used to calculate BL.
Additionally, since the actual electron transport rate (i.e.
fluorescence-derived J) is tightly coupled to Vc,max (Galmes
et al., 2007b) and should indeed reflect gross photosynthesis
(Genty et al., 1989; Valentini et al., 1995), calculations of
BL were confirmed using J directly instead of Vc,max as
a surrogate for leaf biochemistry, to account for possible
errors in the determination of gm and Vc,max (as Vc,max
values are derived from the same AN–Cc curves as gm and
depend on the validity of Rubisco kinetics as estimated by
Bernacchi et al., 2002).
In the current study, the maximum assimilation rate,
concomitantly with gs, gm, and Vc,max, was generally
reached under well-watered conditions; therefore, the control treatment was used as a reference. However, since AN
of irrigated plants declined during the experiment, particularly in spring, presumably due to leaf ageing, the values for
irrigated plants for each day were considered as the
reference for the stressed or recovering plants determined
during the same day. In doing so, photosynthesis limitations
due to leaf ageing were eliminated and, hence, ‘pure’ water
stress limitations were obtained for stressed plants. Whenever one of the involved parameters (gs, gm, and Vc,max) was
higher in stressed than in irrigated plants, its corresponding
Role of mesophyll conductance during water stress and recovery in tobacco | 2383
limitation was set to zero, and the other limitations recalculated accordingly.
Finally, Grassi and Magnani (2005) defined a fourth
photosynthesis limitation associated with leaf temperature
(TL). However, this is not considered here because: (i)
photosynthesis limitations were calculated separately for
each of the three experiments, and the differences in leaf
temperature between irrigated and water-stressed plants
were small (Table 1); (ii) leaf temperature was already
Table 1a. Climatic parameters monitored during the experimental
periods
Maximum and mean daytime (9:00–18:00 h) photosynthetic photon
flux density (PPFDmax; PPFDday), minimum, mean daytime (9:00–
18:00 4), and maximum air temperature (Ta,min, Ta,day, and Ta,max), as
well as the daily sum of evapotranspiration (ET) were determined
from a meteo station at the field site (measuring integrals of 5 min)
and with a portable datalogger (testo 175-H2, Testo AG, Germany).
PPFDmax, PPFDday, Ta,min, Ta,day, Ta,max and ET are means and
standard errors of at least 16 d.
Parameter
Outside
summer
Outside
spring
Inside growth
chamber
PPFDmax (lmol
photons m2 s1)
PPFDday (lmol
photons m2 s1)
ET (mm)
Ta,max (C)
Ta,day (C)
Ta,min (C)
1930
1605
650*
1434674
10746102
600612*
4.560.2
35.7
27.060.3
15.3
3.360.3
28.1
20.160.5
10.7
3.860.1
26.6
25.960.2
21.6
* Despite constant irradiance, small variations of PPFDs were due
to small differences in the incident light, which was measured with
a portable light sensor at the level of leaves used for measurements.
Table 1b. Mean values and standard errors of climatic parameters during the gas-exchange measurements for control and
water-stressed plants
Means and standard errors of at least 16 d (>4 plants per treatment)
are shown, where Ta, and Tl denote mean midday (10:00–14:00 h)
air temperature and leaf temperature, respectively. During all gasexchange measurements, the relative humidity of ambient air was
recorded and the PPFD was kept at 1500 lmol m2 s1, while the
temperature of the leaf chamber (block temperature) was set to 30,
28, and 25 C in the summer, spring, and growth chamber experiments, respectively.
Parameter
Treatment
Outside
summer
Outside
spring
Inside
growth
chamber
PPFD
(lmol m2 s1)
Relative
humidity (%)
Ta (C)
Tl (C)
Ta (C)
Tl (C)
All
1500
1500
1500
All
56.868.4
39.763.1
42.662.1
Control
Control
Stress
Stress
30.460.1
31.060.2
30.560.1
31.460.3
27.560.1
26.760.3
27.660.1
27.860.2
24.960.1
24.560.3
24.960.1
25.960.3
considered in determining gm and Vc,max; and (iii) Grassi
and Magnani (2005) already showed that TL was generally
negligible, reaching maximum values as low as 4–7% even
for leaf temperature differences between the reference value
and the treatment of up to 20 C.
Results
In order to distinguish between the three experimental
conditions, various climatic parameters were monitored
(Table 1). As expected, the highest daily means and
maximum values of PPFD as well as evapotranspiration
(ET), and air and leaf temperature were found during the
summer experiment. The PPFD and ET during spring were
>30% lower than in summer, but the PPFD was still ;50%
higher than in the growth chamber. Maximum air temperature, however, was similar in the growth chamber and
outside during spring (Table 1a), while daily mean temperatures were more similar between summer and growth
chamber conditions. Differences in relative humidity during
the measurements were highest outside during summer
(;60%) and lowest in spring (40%), whereas intermediate
values were observed in the growth chamber (Table 1b).
Among control and stressed plants, leaf and air temperature
differed only a little, although the highest values were
measured in summer plants and the lowest in growth
chamber plants. Overall, the most pronounced differences
among the three experimental periods were observed in
PPFD and leaf temperature.
Subjecting tobacco plants to water stress by withholding
water resulted in non-significant changes of the RWC of
leaves at midday (RWCMid) in the summer experiment
(Fig. 2a). In contrast, RWCMid declined from 65% to 43%
under severe water stress in plants of the spring experiment,
although it was immediately restored to control values
within the first day of re-watering (Fig. 2b). In the growth
chamber experiment, RWCMid decreased slightly during
severe water stress, from initially 78% to 61%, and it was
also completely restored within 1 d of re-watering.
The substrate water availability was reduced after withholding water in all three experiments, reaching ;65, 50,
and 60% of the values for irrigated plants in summer,
spring, and growth chamber, respectively (Fig. 2d–f). The
speed of water stress development differed among the three
experiments. The desired stomatal conductance defining
severe water stress conditions was reached after 11, 8, and
5 d in summer, spring, and growth chamber experiments,
respectively. Differences in water stress progression might
be related to the prevailing climatic conditions, particularly
irradiance and temperature (Table 1), but also to different
pot sizes (pots for spring and summer experiments were
slightly larger than those for the growth chamber).
After withholding water from plants, AN decreased progressively in all three experiments, reaching minimum
values of <5 lmol CO2 m2 s1 during severe water stress
(Fig. 3a–c). AN remained almost unaltered during the
2384 | Galle et al.
Fig. 2. Changes of relative water content around midday (RWCMid) and substrate water availability (as a percentage of controls) in
stressed plants throughout the experiments in summer, spring, and growth chambers. Boxes above the graphs indicate phases of stress
imposition (S), drought acclimation (A), and re-watering (R), while the vertical dashed lines indicate the beginning of the drought
acclimation phase and the beginning of re-watering. Data points represent means and standard errors of at least four replicates.
acclimation phase of severe water stress. The restoration of
AN to control values was reached after subsequent rewatering for 1, 3, and >6 d under growth chamber, spring,
and summer conditions, respectively. AN of daily irrigated
plants (control) remained mostly unchanged throughout the
summer experiment (Fig. 3a), while in the other two cases
(Figs. 3b, c) a decline with time was observed (presumably
due to the ageing of leaves). Slightly different levels of
maximum AN were observed in the three experiments, with
plants growing in summer, spring, and growth chambers
showing values of 21, 25, and 28 lmol m2 s1, respectively.
Due to the water shortage, gs was reduced, resulting in
a decline from ;0.2 mol CO2 m2 s1 in all three experiments to <0.05 mol m2 s1 during severe stress (Fig. 3d–f).
After re-watering gs increased again, reaching (almost)
control levels by the end of the experiments. Overall,
changes in gs and AN followed almost the same trend,
indicating a close co-regulation of both processes.
Although the gm in summer and spring experiments
followed a similar course to gs, its behaviour was different in
the growth chamber experiment (Fig. 3g–i). Starting with
very similar values under control conditions in all three cases,
gm dropped below 0.05 mol m2 s1 during severe water
stress in summer and spring (Fig. 3g, h), whereas it kept
above 0.1 mol m2 s1 during all the experiment in the
growth chamber (Fig. 3i). In fact, gm initially decreased in
parallel with AN and gs under water stress, but then increased
again during the water stress acclimation phase, reaching
values similar to irrigated plants. gm oscillated around the
control values with an up and down course during the stress
adaptation phase, which most probably was related to
similar but smaller alterations in gs. Notably, the differences
in gm between stress and control plants were highest in
summer, where the restoration to control values was also
still incomplete after 6 of re-watering. As compared with
summer, in spring gm declined only later during water stress
imposition, and its recovery was fast (1–2 d) after re-watering
(Fig. 3h).
According to the initial situation of AN, the electron
transport rates (J) were highest in the growth chamber and
lowest in summer (Fig. 3j–l). Moreover, changes in J
followed the time course of AN in all experiments, showing
a decrease of ;20–40% of the control values during severe
water stress and a recovery of the same velocity as observed
for AN. Control plants displayed rather constant values of
J, with the same trends as already observed for AN in spring
and growth chamber (see above).
Alterations of the maximum velocity of carboxylation
(Vc,max) due to water stress were non-significant (Fig. 3m–o),
indicating that the functionality of Rubisco was largely preserved. Also the functionality of the photosynthetic apparatus was preserved throughout the experiments in stressed and
control plants, as indicated by unchanged pre-dawn values of
the maximum quantum efficiency of PSII (Fv/Fm, data not
shown).
When plotting all AN (consisting of control, water stress,
and re-watering data) against the corresponding calculated
CO2 concentration at the sites of carboxylation in the
chloroplasts (Cc), highly significant relationships were
obtained pooling irrigated and water stress data together,
although three different functions were derived for the three
different experiments (Fig. 4a). The steepest slope was
determined for the growth chamber data set, which,
however, resulted in the lowest Vc,max (Fig. 3) due to lower
Role of mesophyll conductance during water stress and recovery in tobacco | 2385
Fig. 3. Changes of leaf parameters related to photosynthetic CO2 assimilation during the experimental periods in stressed (open
symbols) and control (filled symbols) plants. AN, gs, gm, J, and Vc,max denote net photosynthesis rate, stomatal conductance for CO2,
mesophyll conductance for CO2, electron transport rate, and maximum velocity of carboxylation, respectively. Boxes above the graphs
indicte phases of stress imposition (S), drought acclimation (A), and re-watering (R), while the vertical dashed lines indicate the beginning
of the drought acclimation phase and the beginning of re-watering. Data points represent means and standard errors of at least four
replicates.
2386 | Galle et al.
leaf temperature. Under water stress (threshold of AN
indicated by the dashed line), the differences between
experiments were less clear. In contrast to these AN–Cc
relationships, plotting AN on J values resulted in almost
similar slopes of linear regression for all three experiments
(Fig. 4b). A similar relationship could be observed from
AN–gm plots (Fig. 4c), where spring and summer data
revealed the same slopes of regression lines, but growth
chamber data did not correlate well linearly.
Quantitative limitation analysis of photosynthesis underlined the above-described changes during water stress
and recovery after subsequent re-watering. Either Vc,max(Fig. 5a–c) or J- (Fig. 5d–f) based limitation analysis
revealed a very similar picture for the limitations caused by
stomatal (SL) and mesophyll conductance (ML), with
a smaller contribution of bio-/photochemical limitations
(BL) in the Vc,max- than in the J-based analysis. In all three
experiments, SL made up >50% of the total limitation
under severe water stress, while ML accounted for only up
to 20% (except for higher values on the last day of severe
drought in spring). Furthermore, BL did not exceed 10% of
the total limitation. As already observed for the AN, gs, and
gm data during stress and recovery, almost no limitation by
ML and BL was set to growth chamber plants during water
stress and after re-watering (Fig. 5c, f), leading to the most
rapid photosynthetic recovery of all three experiments (only
slightly affected by some lasting SL). Limitation of photosynthetic recovery of the spring plants was mainly driven by
a still high SL and somewhat lower ML and BL (Fig. 5b, e).
The delayed recovery of photosynthesis in the summer
plants was mainly due to a maintained high proportion of
ML (and BL; Fig. 5d) during several days of re-watering
(Fig. 5a, d), while SL contributed only partially to the total
limitation in the initial phase of re-watering.
Fig. 4. The relationships between net photosynthesis rates (AN) and chloroplastic CO2 concentrations (Cc), electron transport rates (J),
and mesophyll conductances (gm) derived from data of the whole experimental periods. Diamonds, circles, and triangles denote
summer, spring, and growth chamber data, respectively. Open symbols represent data during severe drought, while filled symbols
represent the other situations (well irrigated, initial phase of drought, and re-watering). Data points represent means and standard errors
of at least four replicates.
Fig. 5. Quantitative limitation analysis of photosynthetic CO2 assimilation during drought stress and subsequent re-watering in summer,
spring, and growth chamber experiments. The shaded areas represent the percentage of stomatal (SL), mesophyll (ML), and biochemical
(BL) limitation based on control values of AN, gs, gm, and Vc,max or J. In the upper panels (a–c) limitation analyses based on Vc,max values
are depicted, while in the lower panels (d–f) they are based on Jmax values. Calculations were done with mean values of at least five
measurements per treatment and day.
Role of mesophyll conductance during water stress and recovery in tobacco | 2387
Discussion
In the present study, the same experimental design was
applied to tobacco plants under three different environmental conditions, consisting of the imposition of a severe water
stress (although at slightly different velocities due to differences in environmental conditions), followed by an acclimation period of 1 week and a recovery after re-watering. The
most important difference between the three experimental
conditions was observed after re-watering: the rate of
photosynthetic recovery was the slowest during summer
and the quickest under growth chamber conditions. Despite
these differences, the maximum velocity of carboxylation
(Vc,max) and the pre-dawn maximum efficiency of PSII
(Fv/Fm; data not shown) remained almost unchanged
throughout all experiments, indicating preservation of the
photosynthetic machinery and a minor relevance of irreversible damage during severe water stress (Reddy et al.,
2004; Galle et al., 2007; Galmes et al., 2007a). Indeed, the
only changes observed in Vc,max consisted of a decreasing
trend during the experiment in both irrigated and waterstressed plants, which was especially evident in the spring
experiment and presumably related to ageing of leaves
(Niinemets et al., 2005).
Withholding water resulted in a closure of stomata, which
was accompanied by a marked decrease of net photosynthesis (AN) in all three experiments (Fig. 3). Throughout the
periods of water stress imposition, stomatal conductance
(gs) and AN followed the same course, indicating a strong
co-regulation between them, which has been shown elsewhere (Medrano et al., 2002). Moreover, stressed plants
outside (spring and summer) displayed a similar course for
mesophyll conductance (gm). These results are in line with
previous studies, where a decrease of gm has been observed
during water stress (Ennahli and Earl, 2005; Grassi and
Magnani, 2005; Monti et al., 2006; Galmes et al., 2007b). In
contrast to these findings, an acclimation of gm was
observed during water stress under growth chamber conditions (Fig. 3i). After an initial decline of gm in parallel
with gs, gm increased again during prolonged water stress,
reaching values similar to control plants. This restoration to
control values during water stress was not accompanied by
increased gs values, indicating an independent regulation of
gm and gs. Nevertheless, restored gm during prolonged water
stress presumably facilitated photosynthetic recovery after
the beginning of re-watering, because AN and J were
immediately restored to control values within the first day,
while it took longer in spring and, especially, in summer
plants. The quantitative limitation analysis also supported
this explanation, as gm (ML) only marginally contributed to
the total limitation of photosynthetic CO2 assimilation (Fig.
5c, f). Following the idea of gm as a major limiting factor of
photosynthetic recovery, reduced gm values during water
stress might lead to a delayed restoration of AN. In fact,
that has been observed in the spring- and summer-grown
plants (Fig. 5). However, in both experiments, very low gm
values were reached during prolonged water stress, but their
rates of photosynthetic recovery differed considerably.
When comparing limitation components quantitatively,
ML of spring plants contributed only little to the total
limitation during recovery, with an overall high contribution of stomatal limitation (SL) throughout the experiment
(Fig. 5b, e). In contrast, almost 50% of the total limitation
during recovery of summer stressed plants was derived by
ML, while already at the beginning of water stress
imposition a large fraction of ML prevailed (Fig. 5a, d).
Moreover, restoration of gm as well as of AN and gs to
control values was still incomplete after 1 week of rewatering, indicating irreversible or slowly reversible changes
at the cellular level. These results also support the importance of gm in restoration of photosynthesis after relief of
stress, although or even despite a large contribution of SL
to the total limitation of photosynthesis. As already indicated by unaltered Vc,max, the contribution of biochemical
limitation (BL) was relatively small during severe water
stress and recovery, not making up more than 10% of the
total limitation (irrespective of J- or Vc,max-based data).
However, the ML of spring and summer plants was very
similar during the initial phase of severe water stress, while
ML in spring plants levelled off very rapidly during rewatering but in summer plants it did not. The most likely
explanation for this discrepancy might be derived from the
prevailing climatic conditions during the three experimental
periods. In this sense, summer plants experienced much
higher light intensities and temperatures (air and leaf)
during the course of the experiment than growth chamber
plants, with spring plants having an intermediate situation
(Table 1). Excessive light and elevated temperatures have
already been shown to exacerbate water stress effects on
light energy dissipation and xanthophyll cycling (Demmig
et al., 1988; Galle et al., 2007; Galmes et al., 2007a),
photoinhibition and photooxidation (Reddy et al., 2004;
Flexas et al., 2006a), and impairment of Rubisco (Zhou
et al., 2007). Here it is shown for the first time that the
water stress-induced down-regulation of gm is also exacerbated by high light and temperature. This is not surprising
since both light (Gorton et al., 2003; Flexas et al., 2007b,
2008) and temperature (Bernacchi et al., 2002; Gorton et al.,
2003; Yamori et al., 2006) alone have been shown to affect
gm. Moreover, although the mechanisms of gm regulation
are not fully understood, for aquaporins at least, several
described regulatory mechanisms can be light dependent,
including direct phosphorylation of aquaporins (Kjellbom
et al., 1999) and pH-dependent gating of aquaporins
(Tournaire-Roux et al., 2003).
Therefore, recovery of photosynthesis in summer might
be delayed by temperature and light effects superimposed
on water stress-induced changes. The signal(s) for a relatively rapid (spring) or slow (summer) response of gm during
re-watering seem to arise from the mesophyll rather than
from CO2 of the substomatal cavities or stomata (Mott
et al., 2008), while its leaf internal regulation still remains
unclear and awaits further research. Nonetheless, adaptation of gm to severe water stress and rapid photosynthetic
recovery under growth chamber conditions seemed to be
facilitated by the absence of additional stress factors such as
2388 | Galle et al.
excessive light and elevated temperature. Also other
climate-related factors such as the morphology of leaves
might have contributed to the differences in gm (Terashima
et al., 2001; Evans et al., 2004; Tholen et al., 2008),
emphasizing the need for further investigations.
In conclusion, mesophyll CO2 diffusion (i.e. gm) of
tobacco plants declined under prolonged water stress in the
field, but the effect was dependent on the prevailing
conditions, ranging from a long-lasting decline even after
re-watering in outdoor plants in summer to a complete
recovery during the water stress acclimation period in plants
in the growth chamber. Therefore, other factors in addition
to water stress, i.e. excessive light and elevated temperature,
seemed to enhance the decrease of mesophyll conductance
and thereby slowed recovery of photosynthesis during rewatering. In summary, the present study strongly reinforces
the important role of gm during recovery from water stressinduced inhibition of photosynthesis, but shows for the first
time that such a role depends on the prevailing environmental conditions.
Acknowledgements
This study was financed by the Spanish Ministry of
Education and Research: projects BFU2005-03102/BFI
‘Effects of drought on photosynthesis and respiration:
acclimation and recovery’ and BFU2008-00274-E/BFI ‘Regulation of mesophyll conductance to CO2 in relation to
photosynthesis and plant respiration’. The authors would
like to thank Pere J Pons and Sebastià Martorell for field
and technical assistance. The UIB central services are kindly
acknowledged for providing the MS facilities. AG had
a fellowship from the Swiss National Science Foundation.
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