Journal of Experimental Botany, Vol. 52, No. 359, pp. 1323±1330, June 2001
Limitations to photosynthesis of lettuce grown under
tropical conditions: alleviation by root-zone cooling
J. He1,3, S.K. Lee1 and I.C. Dodd2
1
Natural Sciences Academic Group, National Institute of Education, Nanyang Technological University,
1 Nanyang Walk, Singapore 637 616
2
Department of Botany, The University of Queensland, St Lucia 4072, Australia
Received 4 December 2000; Accepted 7 February 2001
Abstract
Aerial parts of lettuce plants were grown under
natural tropical fluctuating ambient temperatures,
but with their roots exposed to two different
root-zone temperatures (RZTs): a constant 20 8CRZT and a fluctuating ambient (A-) RZT from
23±40 8C. Plants grown at A-RZT showed lower photosynthetic CO2 assimilation (A), stomatal conductance
( gs), midday leaf relative water content (RWC), and
chlorophyll fluorescence ratio FvuFm than 20 8C-RZT
plants on both sunny and cloudy days. Substantial
midday depression of A and gs occurred on both
sunny and cloudy days in both RZT treatments,
although FvuFm did not vary diurnally on cloudy days.
Reciprocal temperature transfer experiments investigated the occurrence and possible causes of
stomatal and non-stomatal limitations of photosynthesis. For both temperature transfers, light-saturated
stomatal conductance ( gs sat) and photosynthetic
CO2 assimilation ( Asat) were highly correlated with
each other and with midday RWC, suggesting that
A was limited by water stress-mediated stomatal
closure. However, prolonged growth at A-RZT reduced
light- and CO2-saturated photosynthetic O2 evolution
(Pmax), indicating non-stomatal limitation of photosynthesis. Tight temporal coupling of leaf nitrogen
content and Pmax during both temperature transfers
suggested that decreased nutrient status caused this
non-stomatal limitation of photosynthesis.
3
Key words: Lactuca sativa L., root-zone temperature,
photosynthetic CO2 assimilation, stomatal conductance,
relative water content.
Introduction
The tropical climate of Singapore, which is non-seasonal,
hot and humid, is not suitable for growing temperate
crops. However, lettuce has been successfully grown in
an aeroponics system by exposing the roots to cool
temperatures (15±25 8C) while shoots were maintained at
hot ambient temperatures (Lee and Cheong, 1996; He
and Lee, 1998a, b). Both the formation of compact heads
and the growth and development of root and shoot were
affected by root-zone temperature (RZT) (He and Lee,
1998a, b). However, the effect of RZT on photosynthesis
of temperate lettuce under tropical conditions has not
been investigated.
Limitations of photosynthesis by supra-optimal RZT
are usually ascribed to stomatal closure (Behboudian
et al., 1994; Du and Tachibana, 1994a) depleting carbon
dioxide in the intercellular spaces and at the chloroplast
level. Such closure may result from water stress, as
supra-optimal RZT can alter the balance between water
uptake by the root system and water loss from the shoot.
Although the roots of aeroponically grown plants are
frequently sprayed with nutrient mist, poor root system
To whom correspondence should be addressed. Fax: q65 896 9432. E-mail: [email protected]
Abbreviations: A, photosynthetic CO2 assimilation; Asat, light-saturated photosynthetic assimilation; A-RZT, ambient root-zone temperature;
A [20 8C-RZT, plants grown initially at A-RZT then transferred to 20 8C-RZT; Ci, internal CO2 concentration; Fo, minimal fluorescence yield of a darkadapted sample; Fm and Fv, maximal and variable fluorescence yield obtained from a dark-adapted sample upon application of a saturating light pulse;
FvuFm, dark-adapted ratio of variable to maximal fluorescence (the maximal photosystem II quantum yield without actinic light); gs, stomatal
conductance; gs sat, stomatal conductance measured at a light intensity saturating for photosynthetic assimilation; Lp, root hydraulic conductivity;
Pmax, light- and CO2-saturated photosynthetic O2 evolution; PPFD, photosynthetic photon flux density; RZT, root-zone temperature; RWC, relative
water content; 20 8C-RZT, root-zone temperature of 20"2 8C; 20 8C [A-RZT, plants grown initially at 20 8C-RZT then transferred to A-RZT; VPD,
vapour pressure deficit.
ß Society for Experimental Biology 2001
1324
He et al.
development at high RZT (He and Lee, 1998a, b) may
cause transient water de®cits.
Severe water de®cits (relative water content -70%)
may also reduce photosynthesis by non-stomatal mechanisms, as diagnosed by decreases in the chlorophyll
¯uorescence ratio FvuFm, or decreased light- and CO2saturated photosynthetic O2 evolution (Pmax) (Kaiser,
1987). Such non-stomatal effects are exacerbated by additional stresses such as high temperature and high irradiance (BjoÈrkman and Powles, 1984) or nutrient de®ciency
(Verhoeven et al., 1997). Plants grown at high root temperatures often show decreased nutrient concentrations
(Du and Tachibana, 1994b).
The aims of this study were to determine whether
photosynthesis of plants grown at high RZTs was limited
by stomatal or non-stomatal factors, and to determine
whether water de®cit or nutrient stress could account
for these limitations. Leaf relative water content and
nitrogen content were measured as they closely correlate
with photosynthetic capacity (Kaiser, 1987; Field and
Mooney, 1986). Since photosynthesis of plants grown
for prolonged periods at supra-optimal RZT may be
co-limited by both water and nutrient stresses, reciprocal RZT transfer experiments investigated the timing and
impact of these stresses on stomatal and non-stomatal
limitation of photosynthesis. The effect of diurnal variations in PPFD on photosynthesis of plants at different
RZTs was ®rst assessed on days of differing PPFD.
Understanding the causes of photosynthetic limitation in
plants at different RZTs will assist in management procedures aimed at reducing the root cooling requirements
of temperate crops in the tropics.
Materials and methods
Plant materials and cultural methods
Butterhead lettuce (Lactuca sativa L. cv. Palma) seeds were
germinated on moist Whatman ®lter papers (No. 3) in Petri
dishes. After 3 d, seedlings were transplanted onto polyurethane cubes soaked with water and placed in trays in the
greenhouse. After two more days to allow seedling establishment, they were transplanted to aeroponics troughs (Lee,
1993) in a greenhouse at Nanyang Technological University,
Singapore. The top of each trough was insulated by sheets of
polystyrofoam on which the plants were anchored. Full-strength
Netherlands Standard Composition (Douglas, 1982) nutrient
solution (conductivity ˆ 2.2 mS) was used. Details of the root
temperature control system have been previously published
(Lee, 1993).
Reciprocal transfer between RZTs
RZT transfer experiments were conducted 3 weeks after
transplanting and all measurements were made over the ensuing
10 d. Half the plants were maintained at their original RZT
(either 20 8C-RZT or A-RZT), and the other half were
transferred to the other RZT at 07.00 h. There were thus four
RZT treatments: 20 8C-RZT, A-RZT, 20 8C[A-RZT and
A[20 8C-RZT. The RZT transfer experiment was performed
twice at different times of the year with similar results; results
are presented from only one experiment. All leaf measurements
used the fourth leaf from the base.
Measurement of environmental variables and
photosynthetic parameters in vivo
Three weeks after transplanting, diurnal changes in A and gs
were measured in the greenhouse for three clear sunny and
three cloudy days between 07.00 h and 19.00 h with a portable
open system gas analyser (CIRAS-1, PP-system, Hitchin,
Herts, UK). A leaf area of 2.5 cm2 was used to determine
A and gs under prevailing solar radiation. PPFD and ambient
and leaf temperatures were also measured concurrently with the
quantum sensor and thermistors on the CIRAS-1.
In the RZT transition experiments, light-saturated photosynthetic CO2 assimilation (Asat) and stomatal conductance
(gs sat) of attached leaves were measured simultaneously
between 10.30±11.00 h in the greenhouse with a PPFD of
1350 mmol m±2 s±1 supplied from a metal halide lamp (Wotan
12 V 100 W bulb). Light response curves of both CO2 ®xation and O2 evolution (measured in the laboratory at 25 8C
at saturating CO2 conditions) had previously established
that a PPFD of 1350 mmol m 2 s 1 was saturating for lettuce
leaf photosynthesis (data not shown), as in other studies
(Caporn, 1989).
Measurement of photosynthetic oxygen evolution
on detached leaves
To determine photosynthetic capacity (Pmax) in the absence of
stomatal limitation, ®ve leaves were harvested from ®ve different plants between 11.00±11.10 h and brought back to the
laboratory. Leaves were then kept in a tray of distilled water
under a PPFD of 1350 mmol m 2 s 1 for 30 min. Rates of
maximum photosynthetic O2 exchange were determined with
a leaf disc O2 electrode (Hansatech, King's Lynn, UK) under
a PPFD of 1350 mmol m 2 s 1 at 25 8C at saturating CO2 conditions (1% CO2 from a 1 M carbonateubicarbonate buffer,
pH 9) as described earlier (Ball et al., 1987).
Measurement of chlorophyll fluorescence
All measurements of chlorophyll ¯uorescence were made with
a portable ¯uorometer (PAM-2000, Walz, Effeltrich, Germany)
on attached leaves, immediately following the gas-exchange
measurements. A personal computer equipped with DA-2000
software (Walz) was used for data acquisition. To measure
FvuFm ratio, DLC-8 aluminum leaf clips (2 cm diameter, Walz)
were used to pre-darken (15 min) leaves prior to measurement
and to enable vertical positioning of the PAM ®bre optics
with respect to the leaf surface. Leaves from both 20 8C-RZT
and A-RZT plants were tested for dark adaptation by using
different dark periods from 5±30 min (5 min intervals). The
recovery of FvuFm in light-grown leaves after exposure to darkness for various periods showed that a 15 min dark adaptation was adequate (data not shown). After dark adaptation for
15 min, leaves were initially exposed to a weak measuring beam
to estimate minimal ¯uorescence (Fo), then a 0.8 s saturation
pulse (3000 mmol m 2 s 1) to obtain maximal ¯uorescence (Fm).
Preliminary experiments measured Fm with both 3000 and
6000 mmol m 2 s 1 pulses, and found no effect of the intensity
of the pulse (data not shown). Therefore, a saturating pulse
of white light of 3000 mmol m 2 s 1 was used.
Photosynthesis and root-zone temperature
1325
Measurement of leaf relative water contents
Ten leaf discs of 1 cm diameter were cut and immediately
weighed with an analytical balance for the ®eld fresh weight.
The leaf discs were then ¯oated on distilled water in the dark
for 24 h to determine their turgid weight. Dry weights were
obtained after wrapping the leaf discs in aluminium foil
and oven-drying at 80 8C for 48 h. RWC was determined
as: (fresh weight oven dry weight)u(turgid weight oven dry
weight) 3 100.
Measurement of leaf nitrogen content
Dried leaf samples were weighed and placed in a digestion
tube with Kjeldahl tablet and 5 ml of concentrated sulphuric
acid (H2SO4) (Allen, 1989). The mixture was heated at 250 8C
until it turned clear, then total N content was determined using
a Kjeltec Auto 1030 Analyser.
Statistical analysis
Differences between RZT treatments were discriminated using
Dunnett's procedure at P-0.05. The signi®cance of correlations between plant and environmental variables was tested in
JMP In (SAS Institute Inc., Cary, NC, USA).
Results
Photosynthesis, stomatal conductance and
water relations during a sunny and a cloudy day
On a clear and hot day, ambient greenhouse PPFD
and temperature reached maxima of 1800 mmol m 2 s 1
and 38 8C, respectively (Fig. 1a, b). Leaf temperatures of
20 8C-RZT plants during the middle of the day were
2±4 8C lower than A-RZT plants (Fig. 1b). Maximum
leaf temperatures of 20 8C-RZT and A-RZT plants were
40.6 8C and 42.8 8C, respectively, at 13.00 h. Although
A, gs and FvuFm were lower in A-RZT plants, the diurnal
patterns of these variables were similar at both RZTs,
with substantial midday depression occurring (Fig. 1c, d, e).
There was no diurnal change in Fo at either RZT
(data not shown). Mid-morning maxima and subsequent
declines of A and gs occurred c. 2 h earlier in A-RZT
plants. In plants at both RZTs, A correlated very well
with gs (cf. Fig. 1c, d).
Following three consecutive bright sunny days,
diurnal changes in environmental and plant variables
were also measured on a cloudy day (Fig. 1f±j). Ambient
greenhouse PPFD and temperature reached afternoon
maxima of 230 mmol m 2 s 1 and 35 8C, respectively
(Fig. 1f, g). Leaf temperatures of 20 8C-RZT plants were
no more than 2 8C lower than A-RZT plants. Although
gs showed a similar diurnal pattern to that seen on
sunny days (Fig. 1i), A remained relatively stable from
10.00±16.00 h (Fig. 1h). No diurnal variation of FvuFm
was seen (Fig. 1j). Again, A, gs and FvuFm were lower in
A-RZT plants.
Fig. 1. Diurnal changes in photosynthetic photon ¯ux density (PPFD)
(a, f ), air (solid line, no symbol) and leaf temperature (b, g), net
photosynthetic CO2 assimilation (A) (c, h), stomatal conductance (gs)
(d, i), and FvuFm (e, j) in lettuce plants grown under 20 8C-RZT (k) and
A-RZT (m) on a representative sunny (a±e) and a representative cloudy
(f±j) day. Each point is the mean"standard error of ®ve measurements
on leaf 4 from ®ve different plants.
The extent of midday depression of A and gs on three
sunny (maximum PPFD c. 1800 mmol m 2 s 1) and three
cloudy (maximum PPFD-250 mmol m 2 s 1) days is
summarized (Table 1). On sunny days, midday depression of A and gs were similar in magnitude, and 20±30%
greater in A-RZT plants. On cloudy days, the midday
depression of A (30%) was much less than the depression
of gs (70%). RZT had no effect on the magnitude of
midday depression of A and gs on cloudy days.
Leaf RWC of A-RZT plants was lower than 20 8CRZT plants on any measurement occasion (Table 2).
Pre-dawn RWC was 3±6% and 29% higher than midday
RWC in 20 8C-RZT and A-RZT plants, respectively. The
lower pre-dawn RWC of A-RZT plants than 20 8C-RZT
1326
He et al.
Table 1. Maximum and minimum A and gs and midday depression of A (mmol m
at 20 8C- and A-RZT
2
s 1) and gs (mmol m
2
s 1) of lettuce plants grown
Each value is the mean"standard error of 15 measurements made on three sunny and three cloudy days.
Weather
RZT
Maximum A
(10.00±12.00 h)
Minimum A
(12.00±15.00 h)
Midday depression
of A (%)
Maximum gs
(10.00±12.00 h)
Minimum gs
(12.00±15.00 h)
Midday depression
of gs (%)
Sunny day
20 8C-RZT
A-RZT
20 8C-RZT
A-RZT
19.4"1.3
4.9"0.7
9.5"0.7
3.7"0.2
6.5"0.8
0.6"0.1
6.5"0.3
2.6"0.1
67"2
88"1
31"4
29"5
897"46
354"17
518"6
159"10
397"26
38"5
151"13
42 3 7
58"1
89"1
71"2
73"5
Cloudy day
Table 2. Leaf RWC (%) measured at pre-dawn and midday of
lettuce plants grown at 20 8C- and A-RZT
PPFD at midday was 1580 (sunny day) and 200 (cloudy day)
mmol m 2 s 1, respectively. Each value is the mean"standard error of
®ve measurements.
RZT
RWC (%)
Sunny day
20 8C
Ambient
Cloudy day
Pre-dawn
Midday
Pre-dawn
Midday
95.1"0.2
88.8"0.4
89.2"0.3
60.1"0.5
95.2"0.2
89.1"0.2
92.1"0.3
75.9"0.4
plants suggested some residual water stress. However,
¯oating A-RZT plants on a beaker of nutrient solution in
a water-saturated atmosphere in the laboratory also gave
a similar RWC (data not shown). In both determinations,
the leaf discs changed colour when ¯oated on distilled
water to determine their turgid weight, suggesting water
in®ltration of air spaces. Attempts at removing this water
by vacuum prior to the measurement of turgid weight were
unsuccessful (the vacuum also removed cellular water
from the discs resulting in turgid weights which were
less than the fresh weights). Leaves of A-RZT plants have
a greater volume of air spaces than 20 8C-RZT plants
(Flanigan, 1999) and thus the relative increase in
turgid weight will be greater. The difference in pre-dawn
RWC between RZT treatments therefore re¯ects differences in leaf anatomy, rather than the development of
water stress in the A-RZT plants. Measurement of cellular turgor using the pressure probe will be necessary to
con®rm this.
Experiments of reciprocal transfer between RZTs
Fig. 2. Changes in midday PPFD (a), RWC (b) and nitrogen content (c)
of leaf 4 of lettuce plants grown and maintained at 20 8C-RZT (k) and
A-RZT (m) and those grown at 20 8C-RZT but transferred to A-RZT
(n) and those grown at A-RZT transferred to 20 8C-RZT (m). All
measurements were made between 12.30±13.00 h. Each point is the
mean"standard error of ®ve measurements.
Midday leaf RWC did not ¯uctuate with prevailing PPFD
(Fig. 2a, b). Average RWC of 20 8C-RZT and A-RZT
plants were 90% and 53%, respectively (Fig. 2b). In
20 8C[A-RZT plants, RWC decreased from the ®rst day
of RZT transfer (P-0.05) and continued to decrease
throughout the experiment. RWC of A[20 8C-RZT
plants did not change for 3 d after RZT transfer,
although RWC had increased to 87% after 10 d
(Fig. 2b). Both sets of transferred plants showed a similar
RWC 5 d after transfer.
Leaf N content was 32% lower in A-RZT plants
than 20 8C-RZT plants (Fig. 2c). While leaf N content
remained stable in plants maintained at the one RZT,
transfer of plants between RZTs altered N content after
6 d. At the end of the experiment, A[20 8C-RZT plants
Photosynthesis and root-zone temperature
and 20 8C[A-RZT plants had a similar leaf N content
(Fig. 2c).
To prevent changes in PPFD obscuring any
acclimation of plants to altered RZT during the 10 d
period, all photosynthetic measurements (gs, A, Pmax)
were performed under light-saturating (PPFD ˆ
1350 mmol m 2 s 1) conditions. Plants were maintained
in the greenhouse at different RZTs while gs sat and Asat
were measured. Plants maintained at the one RZT
showed constant gs sat and Asat of attached leaves,
with both parameters being c. 70% lower in A-RZT
plants (Fig. 3a, b). When plants were transferred from
20 8C-RZT to A-RZT, gs sat and Asat signi®cantly
(P-0.05) decreased from the ®rst day of RZT transfer.
gs sat and Asat of A[20 8C-RZT plants was similar to
A-RZT plants during the ®rst 3 d post-transfer, and then
gradually increased. Parity of Asat and gs sat in transferred
plants was reached 5 d after transfer.
Light- and CO2-saturated photosynthetic O2 evolution
(Pmax) of detached leaves from plants remaining at one
Fig. 3. Changes in light-saturated (PPFD of 1350 mmol photons
m 2 s 1) stomatal conductance (gs sat) (a) and photosynthetic CO2
assimilation (Asat) (b) of attached leaves; and light- and CO2-saturated
photosynthetic O2 evolution (Pmax) (c) of detached leaves of lettuce
plants grown and maintained at 20 8C-RZT (k) and A-RZT (m) and
those grown at 20 8C-RZT but transferred to A-RZT (n) and those
grown at A-RZT transferred to 20 8C-RZT (m). All measurements were
made between 11.00±11.30 h. Each point is the mean"standard error
of ®ve measurements on leaf 4 from ®ve different plants.
1327
RZT was also constant during the 10 d period, with Pmax
of A-RZT plants 37% lower than 20 8C-RZT plants
(Fig. 3c). Pmax did not signi®cantly (P-0.05) decrease
during the ®rst 4 d post-transfer in 20 8C[A-RZT
plants, although it declined by 22% after 10 d. This
decrease was much less than the decreases in Asat measured on attached leaves in the greenhouse (Fig. 3b).
In A[20 8C-RZT plants, Pmax gradually increased
from 3 d post-transfer and was statistically equivalent
to 20 8C-RZT plants 10 d after the RZT transfer. Parity
of Pmax in transferred plants was reached 8 d after
transfer.
Discussion
Although the shoot environment of all plants was
identical, cooling the roots to 20 8C-RZT allowed higher
assimilation rates by reducing both photoinhibition and
stomatal closure.
On cloudy days, there was no dynamic high PFFDinduced photoinhibition at either RZT. However, chronic
photoinhibition of A-RZT plants was indicated by their
lower FvuFm (Fig. 1j) and decreased chlorophyll content
(data not shown) compared to 20 8C-RZT plants. Such
chlorophyll loss seems to be a photoprotective strategy
to reduce light absorption (Verhoeven et al., 1997) rather
than high light-induced chlorophyll oxidation, since no
increase in Fo (characteristic of photodamage) (Osmond,
1994) was detected.
On sunny days, high PPFD±induced dynamic photoinhibition was entirely attributable to decreased Fm. The
relative changes in FvuFm were less than the relative
changes in A (Fig. 1c, e) suggesting that although
photoinhibition contributed to the midday depression
of A, there was an additional effect of stomatal closure.
Studying the dynamic responses of A and gs to
environmental perturbations may help separate the interrelationships between A and gs (Jones, 1998). Irrespective of RZT, diurnal studies showed highly signi®cant
(P-0.05) correlations between A and gs on both cloudy
and sunny days. However, there was a clear example
where the diurnal responses of A and gs were de-coupled.
Between 10.00 h and 16.00 h on cloudy days, plants at
both RZTs showed substantial midday stomatal closure
which was unaccompanied by reductions in A (cf. Fig. 1h,
i), indicating that stomatal behaviour was independent
of A at low PPFD.
Pronounced midday stomatal closure occurred in
20 8C-RZT plants (Fig. 1d, i) despite midday leaf RWC
being 90% (Fig. 2b). This would seem to exclude leaf
water de®cit as an explanation, although localized water
de®cit related to high transpiration rates could still occur.
The importance of temperature and VPD on the relative
magnitude of midday stomatal closure varied with
RZT. In 20 8C-RZT plants, the greater magnitude of
1328
He et al.
midday stomatal closure on cloudy days than sunny days
(Table 1) seems inconsistent with the lower temperature
and VPD on cloudy days. In A-RZT plants, the higher
leaf temperatures (Fig. 1b) and greater midday stomatal
closure (Table 1) on sunny days suggested that temperature (or some co-variate such as higher VPD or
increased ABA delivery to the stomata) had an additional
effect on midday stomatal closure. Separating the effects
of temperature and VPD on midday stomatal closure
would require controlled environment facilities to examine the effects of one variable while keeping the other
constant (Roessler and Monson, 1985).
The greater midday stomatal closure of A-RZT
plants on sunny days is characteristic of droughted
plants (Tenhunen et al., 1981). That shoot water de®cits
occurred in A-RZT plants is indicated by their very low
midday leaf RWC (Fig. 2b), even though root water
contents remained greater than 94% (data not shown)
due to frequent misting of the root system with nutrient
solution. Leaf RWC recovered overnight (Table 2), suggesting that transient water de®cits occurred during
the day in A-RZT plants due to a diurnal imbalance
of transpiration and root water uptake. Temperatureinduced alterations in root morphology (He and Lee,
1998a) are likely to decrease root hydraulic conductivity
(Lp) (Dodd et al., 2000), resulting in the development of
water stress under high transpiration rates in the middle
of the day. The transfer experiment showed that RZTinduced differences in midday RWC were readily reversible over a 10 d period (Fig. 2b); presumably due to
reduced root hydraulic conductivity (Lp) in 20 8C[
A-RZT plants (Dodd et al., 2000) and the initiation of
new roots in A[20 8C-RZT plants (LP Tan, J He and
SK Lee, unpublished observations). The contributions of
RZT-induced anatomical and physiological alterations
in Lp would merit future investigation in this aeroponics
system, especially given the apparent dependence of
lettuce shoot physiology on RWC (Fig. 4).
The highly signi®cant (P-0.05) correlations between
gs sat and RWC in both temperature transfers (Fig. 4a)
suggested that stomatal closure was directly caused by
reduced RWC. In contrast, lettuce grown in soil columns
where the upper layer was allowed to dry showed up to
a 90% decline in gs while RWC changed by only 5%
(Gallardo et al., 1996), suggesting additional effects of
a root-derived chemical signal such as ABA (Davies and
Zhang, 1991).
The close correlation between gs sat and Asat in plants
transferred between RZTs suggests a stomatal limitation
of photosynthesis. Stomatal closure decreases intercellular CO2 concentration (Ci) which then limits A. However,
non-uniform stomatal closure under drought could overestimate Ci, thus suggesting non-stomatal limitation of
photosynthesis when none existed (Farquhar et al., 1987).
The possibility of non-uniform stomatal closure in
Fig. 4. Light-saturated (PPFD of 1350 mmol photons m 2 s 1) stomatal
conductance (gs sat) (a) photosynthetic CO2 assimilation (Asat) (b) of
attached leaves; and light- and CO2-saturated photosynthetic O2
evolution (Pmax) of detached leaves (c) plotted against midday RWC.
Plants were grown and maintained at 20 8C-RZT (k) and A-RZT (m)
or grown at 20 8C-RZT but transferred to A-RZT (n) and grown at
A-RZT transferred to 20 8C-RZT (m). Data are from Figs 2 and 3.
Data for plants maintained at one RZT are averages from 11 d of
measurement. Error bars have been omitted for clarity.
20 8C[A-RZT plants cannot be excluded, as it occurred
in droughted Phaseolus vulgaris plants subjected to a
similar (although slower developing) water de®cit
(Sharkey and Seeman, 1989). Without knowing whether
stomatal closure was uniform (which would validate
measurement of Ci), an indirect method of determining
non-stomatal limitation of photosynthesis (measuring
photosynthesis under saturating CO2; Fig. 3c) was
preferred.
Previous investigators have generally attributed negative effects of high RZT on A to stomatal closure
(Behboudian et al., 1994). While this was certainly the
initial response of 20 8C[A-RZT plants, a shift to nonstomatal regulation of photosynthesis occurred as time
progressed. This was evident in 20 8C[A-RZT plants as
a divergence from the Asat versus RWC relationship at
RWC-70% (Fig. 4b). More conclusive evidence of nonstomatal limitation of photosynthesis in these plants
was the decline in Pmax measured under saturating CO2
(Fig. 3c). An absence of severe water de®cits in previous
Photosynthesis and root-zone temperature
investigations may account for the lack of reports of
non-stomatal effects of high RZT.
The relationship between RWC and Pmax in 20 8C[
A-RZT plants (Fig. 4c) accorded with that found in slowly
dehydrated detached leaves of mesophytic plants (Kaiser,
1987). However, the lower Pmax of A[20 8C-RZT plants
compared to 20 8C[A-RZT plants at RWC)70%
(Fig. 4c), provided evidence of non-stomatal limitation
of A that was not mediated by water de®cit. It seems
likely that nutrient de®ciencies due to poor root development could be responsible for the declines in Pmax in
A-RZT plants. Parallel analyses of leaf nitrogen content showed tight temporal coupling of leaf N content
(Fig. 2c) and Pmax (Fig. 3c) throughout the reciprocal
temperature transfers. A full evaluation of the role of
N de®ciency in mediating the decline in photosynthesis
of A-RZT plants would require comparison with the
photosynthetic responses of plants grown at 20 8C-RZT
subjected to N deprivation.
Conclusions
Plants transferred from 20 8C-RZT to A-RZT showed
considerable stomatal limitation of photosynthesis as
leaf RWC declined, with non-stomatal limitation occurring only when leaf nitrogen content decreased. In contrast, plants grown for prolonged periods at A-RZT
showed non-stomatal limitation of photosynthesis which
remained in A[20 8C-RZT plants even as leaf water
relations improved. Future studies should focus on how
A-RZT limits water and nutrient uptake.
Temperature transfer experiments may have practical
signi®cance to tropical aeroponics production if plants
can be established at one RZT and cropped at another,
as a shorter period of time at cool RZTs would reduce
cooling costs. However, this experiment suggests that
unless lettuce plants are cooled to 20 8C-RZT after early
crop establishment, yield penalties will result from the
rapid limitation of photosynthesis.
Acknowledgements
This project was supported by the Academic Research Fund,
Ministry of Education, Singapore.
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