Reduction of leaf photosynthesis due to water stress has been analyzed into various components ... Photosynthesis, transpiration and chlorophyll fluorescence.
Potato Research 32 (1989) 17-32
Effects of water stress on photosynthesis and chlorophyll fluorescence of five potato cultivars A. H. C. M. S C H A P E N D O N K , C. J. T. SPITTERS and P. J. GROOT Foundation for Agricultural Plant Breeding (SVP), P.O. Box 117, 6700 AC Wageningen, the Netherlands Accepted for publication 15 August 1988
Additional key words: drought tolerance, stomatal behaviour, breeding
Summary Reduction of leaf photosynthesis due to water stress has been analyzed into various components and genetic variation in these components has been evaluated. Five potato cultivars were grown on nutrient solution in a conditioned glasshouse. Water stress was imposed by adding polyethylene glycol to the nutrient solution. Photosynthesis, transpiration and chlorophyll fluorescence were measured on intact leaves during the stress period and after recovery from the stress. Water stress reduced photosynthesis, initially as a consequence of stomatal closure, but after 3 days increasingly by inhibiting directly the photosynthetic capacity (mesophyll limitation). Stomatal closure correlated with the reduction in photosynthesis, but it was not the sole cause of this reduction because the internal CO 2 concentration in the leaves was not affected by water stress, indicative of inhibitory factors other than stomatal ones. Chlorophyll fluorescence emission suggested that the Calvin cycle was inhibited, while quantum efficiency was not affected at 17 ~ Increasing the temperature to 27 ~ reduced quantum efficiency but only in the stress environment. The recovery of young leaves after relief of the stress was associated with a lower stomatal conductance but a higher mesophyll conductance compared with the control, which caused a low internal CO, concentration and probably invoked photo-inhibition and leaf damage. Cultivar differences in photosynthetic rate were highly significant under both optimal and stress conditions, and corresponded with differences in mesophyll conductance.
Introduction W a t e r stress is a m a j o r c o n s t r a i n t to w o r l d p o t a t o p r o d u c t i o n . T h e effects o f water stress on t u b e r yield d e p e n d on the a g g r e g a t e r e s p o n s e o f m o r p h o - p h y s i o l o g i c a l processes, such as p h o t o s y n t h e s i s , l e a f area e x p a n s i o n , l e a f senescence, p a r t i t i o n i n g o f a s s i m i l a t e s w i t h i n the p l a n t , t u b e r i n i t i a t i o n a n d b u l k i n g (reviews by Van L o o n , 1981, 1986). It d e p e n d s also on the t i m i n g o f the stress within the growth p e r i o d (Spitters & S c h a p e n d o n k , 1988) a n d on c l i m a t i c a n d soil c o n d i t i o n s . Selection for d r o u g h t tolerance is t h e r e f o r e c o m p l i c a t e d by the m a n y processes involved a n d their i n t e r a c t i o n with the e n v i r o n m e n t . T h e present s t u d y is restricted to the effect o f water stress on the rate o f p h o t o s y n t h e sis u n d e r c o n t r o l l e d c o n d i t i o n s . R e d u c t i o n in p h o t o s y n t h e t i c rate is a n a l y z e d into various c o m p o n e n t s a n d genetic v a r i a t i o n in these c o m p o n e n t s is evaluated. This m a y cont r i b u t e to a m o r e c o n s c i o u s selection o f p a r e n t s for h y b r i d i z a t i o n a n d to the d e v e l o p m e n t o f screening tests for key factors in d r o u g h t tolerance.
~lotato Research 32 (1989)
17
A. H. C. M. SCHAPENDONK, C. J. T. SPITTERS AND P..[. GROOT
In potato, reduced photosynthetic rate due to water stress has often been reported (reviews by Bodlaender et al., 1986; Van Loon, 1986). However, attributing of these effects to the underlying processes o f CO 2 transport from outside the leaf to the site o f carboxylation, electron transport, and CO2 fixation in the Calvin cycle has received little attention. In other plant species, it has been demonstrated that water stress can reduce the photosynthetic rate indirectly by closure o f the stomata or directly by a reduction of the photosynthetic capacity of the leaves. There is, however, no consensus about the primary site of the reduction in photosynthesis (review by Kaiser, 1987). There is also no consensus whether photoreactions in the thylakoid membranes or biochemical reactions of the Calvin cycle are most affected (Keck & Boyer, 1974; Ogren & Oquist, 1985). Furthermore most experiments do not distinguish between water stress and heat stress. These factors are positively correlated but it is desirable to measure their effects separately (Ceccarelli, 1984). Material and methods
Experimental design Water stress was imposed as uniformly as possible over the root system of the various plants by growing the plants on nutrient solution and adding polyethylene glycol to establish a low matrix potential in the root environment. Five potato cultivars were chosen for the experiment on the basis o f their differences in drought tolerance in the field (Beschrijvende Rassenlijst voor Landbouwgewassen; Dutch List of cultivars) and in pot experiments (Beekman & Bouma, 1986): Alpha, Bintje, Saturna, Kennebec and Veenster. Sprouted eye pieces were planted on 20 April 1987 in small tubes filled with rockwool. The tubes were pierced through the bottom of small boxes (35 x 35 z 25 cm), filled with coarse sand, to allow for stolon growth and tuber formation. Each box contained four plants o f the same cultivar and there were three replicates for the control and the drought treatment. The roots grew through the rockwool into containers of 8 1, through which a nutrient solution (Steiner solution half strength) was circulated from two main reservoirs connected to the containers by a system of pipes. The rockwool was in contact with the nutrient solution, thus acting as a wick to keep the water content in the stolon boxes at a constant level. Plants were grown in a glasshouse with air temperatures at 17 ~ (day) and 12 ~ (night). The nutrient solution was kept at 18 ~ Fifty days after planting, on 9 June (day 0) half o f the plants were transferred to containers with nutrient solution and 10 ~ polyethylene glycol (PEG, M = 20,000). The addition o f PEG reduced the water potential in the root zone to - 0.27 MPa (pF = 3.4) by lowering the matrix potential component (Steuter, 1981). The resulting viscosity of the nutrient solutions hampered circulation and therefore oxygen was supplied by a pump. Transpiration by the plants increased the concentration o f PEG slightly. On day 6, water stress was enhanced by doubling the percentage o f PEG, equivalent to a matrix potential o f -1.16 MPa (pF=4.1). On day 8, the vapour pressure deficit of the ambient air was enhanced by a temperature jump from 17 ~ to 27 ~ On day 10, the solutions were replaced by water and the temperature was reset to 17 ~ to allow for recovery. Fig. 1 shows a diagram o f the daily reference evapotranspiration for a short grass cover as a function o f global radiation and temperature, calculated by (a) the Makkink equation (De Bruin, 1988) and (b) the water stress during the measuring period. A time table of the 18
Potato Research 32 (1989)
EFFECTS OF WATER STRESS
(mm/day)
Evspotranspiration
potential (M
Water
2.00
0.00 '
1.60
-0.30
1,20
-0.60
0.80
-0.90
0.40
- 1.20
Ps)
~
a
0.00 0
i 2
4
b
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6
8
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14
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- 1.80
0
I
i
i
i
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i
i
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2
4
6
8
10
12
14
16
Days
Days
Fig. 1. Calculated reference evapotranspiration (a) and the matrix potential in the nutrient solution (b) as a function of time after onset of the stress.
Table 1. Time schedule of the measurements of photosynthesis (P), chlorophyll fluorescence (F) and the osmotic value of cell sap (O) and the leaf number sampled (unfolded from the top of the plant at day 0). On 9 June 16.00 h half of the plants were transferred to containers with nutrient solution and l0 °70 polyethylene glycol. Date
Day hr.
Measurement
9 l0 12 16 17 18 24
0 l 3 7 8 9 15
P; P; P; F P; P;
June June June June June June June
F F O F F
Leaf hr. 7; '7; 7; 7 7, 7,
7 7 7 l; 7, I l; 7, I
m e a s u r e m e n t s is depicted in Table 1.
Photosynthesis and transpiration Gas exchange m e a s u r e m e n t s were carried out o n leaves attached to the plant with a portable leaf c h a m b e r analyzer (LCA; Analytical Development Co. (ADC), UK). T h e measuring leaves (2 top leaflets per pot) were the sixth or seventh unfolded leaves from the top. These leaves were fully expanded and they will be referred to as 'old' leaves.
Potato Research 32 (1989)
19
A, H. C. M. SCHAPENDONK, C. J. T, SPITTERS AND P. J. GROOT
Young leaves, unfolded during the stress treatment and with a m i n i m u m length o f 5 cm are denoted as 'young' leaves. Subsequent measurements were made on the same leaves. Air was obtained from a gas cylinder to ensure constant composition. It was led through a water bath to humidify it before entering the leaf chamber. All measurements were done at light saturation, provided by an incandescent lamp cooled by a fan. Average conditions within the c h a m b e r were: 1700/~mol quanta m - 2 s J (340 W m - 2 ) of photosynthetically active radiation ( 4 0 0 - 7 0 0 rim), temperature of 22.6 ~ vapour pressure deficit of 0.57 kPa, and CO2 concentration of 330 vpm. Rates of photosynthesis and transpiration were calculated from the measured concentrations o f CO 2 and vapour in the ingoing and outgoing air stream and the flow rate of the stream by the procedure described by von C a e m m e r e r & Farquhar (1981). Resistances and conductances were estimated on the basis of the following equations: P
=
(c e -
T = (w e -
ci)/
(Fc, b +
Fc,s) =
wi) / ( r ~ , b + r,,,0
(c i -
Co)/
re. m
(1)
(2)
where P is the gross photosynthetic rate (gCO 2 m -2 h-~); T the transpiration rate (gH20 m -2 h - I ) ; c the CO2 concentration (vpm) with indices e, i and o referring to outside the leaf, inside the substomatal cavity, and at the place of carboxylation, respectively; w the water vapour pressure (kPa) with indices e and i referring to outside and inside the leaf; r the resistance (s m -t) with the first indices c and w referring to CO 2 and water vapour, and the second indices b, s and m to b o u n d a r y layer, stomata and mesophylI, respectively. Mesophyll resistance consists of a small transport c o m p o nent and a dominating carboxylation component. Conductances were calculated as the reciprocals of the corresponding resistances (g= 1/r). External concentrations c e and we were measured. The b o u n d a r y layer resistance (rw,b) was estimated to be 18.6 s m -j from measurements with wet filter paper. Vapour pressure inside the leaf (w 0 was assumed to equal the saturated vapour pressure at the leaf temperature. Leaf temperature was estimated from the energy balance of the leaf according to the method described by G o u d r i a a n (1977, p. 78). Leaf temperature exceeded air temperature by, on the average, 2.5 ~ in the control and 3.5 ~ in the stress environment. Vapour pressure deficit (VPD) across the interface between leaf and air was therefore twice as high as the deficit above the leaf, which emphasizes the importahce of accounting for difference in leaf and air temperature when estimating the resistances. Stomatal resistance for water vapour transport (rw.s) was then estimated from measured transpiration rate from Equation 2. Stomatal resistance for CO2 (re.s) amounts to 1.6 times that for water vapour (r,,.0, and b o u n d a r y layer resistance for CO 2 (re.b) is 1.37 times that for water vapour (r,,..b) (Von C a e m m e r e r & Farquhar, 1981). The CO2 concentration at the site of carboxylation is equal to the compensation point, which was supposed to be 45 vpm. Subsequently, c i and re.m were estimated from gross photosynthesis by Equation 1. Since leaves were exposed to high light for only a short period, gross photosynthesis was derived from the measured rate of net photosynthesis supposing 5 ~ dark respiration. Water use efficiency is defined here as CO 2 uptake per unit o f transpiration ( W U E = P/T, g CO 2 g - l H20)" From Equations 1 and 2 and the ratio o f the diffusion coefficients of CO2 and HzO, water use efficiency, adjusted for the vapour pressure deficit (VPD) gradient (we-wi), is de20
Potato Research 32 (1989)
EFFECTS OF WATER STRESS
rived to approximate: VPD-WUE
= VPD.P/T
= (c e - c i ) / 1.6
(3)
which is constant when ci and c~ are constant.
Osmotic potential Osmotic potentials were determined on companion leaves that were harvested on day 7, at three time intervals: 9.00 am, 12.00 am, 15.00 pm. Companion leaves of three plants per pot (seventh unfolded leaves from the top) were placed on water to restore the potential turgescence and subsequently frozen at liquid nitrogen temperature. Osmotic potentials were determined upon thawing with a Wescor vapour pressure osmometer.
Chlorophyll fluorescence Chlorophyll fluorescence was measured to discriminate between various components of photosynthesis in relation to water stress (Havaux & Lannoye, 1985; Schreiber & Bilger, 1985). Chlorophyll fluorescence was measured on attached leaves by a weak measuring light beam (not photosynthetically active) pulsed with a high frequency (100 kHz), at different wavelengths to the chlorophyll fluorescence. The photodetection system was locked to the frequency of the measuring light beam, thus preventing interference of the measurements by light scattered from the photosynthetically active light (Schreiber et al., 1985). Intact leaves o f both stress and control plants were dark adapted by folding aluminum foil around the leaves, 20 minutes before the measurement. Fluorescence induction curves were recorded using a modulation fluorometer (PAM 101 Chlorophyll Fluorometer, H. Walz, Effeltrich, FRG). The leaf was clamped in a small cuvette, flushed with air. The fluorescence signals curves were related to the efficiency of energy transfer in the chloroplasts and to the mesophyll conductance. The latter comprises the rate of electron transport from photosystem II to photosystem I and the Calvin cycle activity. When photosystem I1 is fully oxidized, fluorescence is low (i.e. quenched). A saturating light flash was fired to determine the maximal fluorescence (Fro). The ratio between Fm and the initial fluorescence after dark adaptation (Fo) is an indication of the efficiency of energy transfer from antennae pigments to photosystem lI. Synchronously, the red light (100 ~mol m -2 s -~) was switched on to activate photosynthesis. Repetitive saturating light flashes (10 000 ;tmol m -z s -t) were applied at a frequency of 0.25 Hz to induce transients, (Fv)~, superimposed on the fluorescence evoked by the red light (Fv). A detailed discussion of the fluorescence quenching analysis is given by Schreiber & Bilger (1985). The ratio of the fluorescence signals just before and during the flashes gives information about the amount of oxidized photosystem II acceptors at that moment. Adopting the proposition of Krause et al. (1982) and Bradbury & Baker (1983), this ratio increases in the light due to the activation of the electron transport chain which causes a re-oxidation of the acceptor site of photosystem II and thus a decrease of the fluorescence (Q-quenching). Additional information, regarding the energy status of the chloroplasts can now be derived from the differences between the maximum fluorescence in the first flash (Fm) and the lower responses in the subsequent flashes (F,)~. The pH-gradient increases
Potato Research 32 (1989)
21
A. H. C. M. SCHAPENDONK,
C . J. T. S P I T T E R S
AND
P. J. G R O O T
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Potato Research 32 (1989)
EFFECTS OF WATER STRESS
initially in the light and is subsequently used for the formation of ATP, which in turn feeds the Calvin cycle with energy. This is reflected by the slow decrease o f the Equenching, when the Calvin cycle is activated.
Results There are two conflicting views on the m a j o r sites for the inhibition of photosynthesis due to water stress; stomatal and mesophyll limitation. The experimental results of the gas exchange measurements wiI[ be interpreted along the lines of these theories.
(1) Stomatal limitation. According to the classical view, water stress induces closure of the stomata, either due to a lowered leaf water content or due to some other signal transduced to the leaves. The greater resistance of the stomata for CO, diffusion results in a reduction of the C O , concentration inside the leaf and so in a lowered rate o f photosynthesis (reviews by Bradford & Hsiao, 1982; Ceccarelli, 1984). This mechanism of stomatal limitation is reflected in a reduced internal CO, concentration (smaller value of the ci/c e ratio) and a greater share of the gas phase resistance (r b + rs) to the total resistance for CO2 (r b + r~ + rm). Photosynthesis is less inhibited than transpiration because the gradient for CO 2 is increased, while that for water vapour remains the same (Equations 1 and 2). Water use efficiency, at given vapour pressure deficit, is therefore enhanced by water stress (Equation 3).
(2) Mesophyll limitation. In addition to stomatal closure, water stress can reduce the photosynthetic capacity directly, either by inhibiting the Calvin cycle or the rate of electron transport over the chloroplast membranes (review by Kaiser, 1987). Stomatal aperture is also reduced, but in such a way that the internal CO: concentration remains unaffected (e.g. Wong et al., 1979, 1985). Thus stomatal aperture adapts to the mesophyll limitation and both show a correlated response to water stress, probably mediated by ABA (Schulze, 1986). The mechanism of mesophyll limitation is expressed in that the q / c e ratio and the share of gas phase resistance to total resistance remain unchanged when water stress occurs. Photosynthesis and transpiration are equally reduced and water use efficiency remains the same. Primary effects o f water stress Measured rates of photosynthesis and transpiration and derived components are presented averaged over the five cultivars (Fig. 2, Table 2) and for each of the cultivars individually (Fig. 3) as a function of time after exposure to water stress. As expected, both the rates of photosynthesis and transpiration dropped after exposure to water stress (Fig. 2a, b). The CO_, concentration in the Ieaf was, however, only reduced at
Fig. 2. Time courses of gas exchange parameters for 'old' leaves (open triangles) and 'young' leaves (closed triangles) in the stress treatment expressed relative to the control, averaged over the five cultivars. Presented are (a) rate of photosynthesis, (b) transpiration rate, (c) product of water use efficiency and leaf vapour pressure deficit, (d) mesophyll conductance, (e) ratio between internal and external CO, concentration, (O share of gas phase resistance to total resistance of CO 2 transport. Bars represent the standard error of difference.
Potato Research 32 (1989)
23
A. H. C. M. S C H A P E N D O N K , C. J. T. S P I T T E R S A N D P. J. G R O O T
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EFFECTS OF WATER STRESS
Photosynthesis
C02inlCO2ex
1.00
1.50
I ..4•
0.50
0.00
/
/
\
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Fig. 3. Time courses of (a) photosynthesis and (b) the ratio of internal and external CO: concentrations in the stress treatment, expressed relative to the control treatment. Cuhivars: Alpha (+), Veenster ( • ), Bintje ( A ), Saturna (+), Kennebec ( o ).
day I and increased to the control level after 3 days (Fig. 2e). This indicates that initially the rate of photosynthesis reduced immediately by stomatal closure but within a few days it was not the only cause of the reduction any longer and the conductances for CO2 in the gas phase and in the mesophyll were equally reduced (Fig. 2f). The mesophyll conductance is determined by the rate o f electron transport (Q-quenching) from photosystem 11 to photosystem 1 over the thylakoid membranes and by the rate o f CO2-assimilation by the Calvin cycle (E-quenching). From the time course of the Q-quenching (Fig. 4a), it may be concluded that the redox-state o f photosystem I1 was not affected by water stress, except for day 9, after the temperature increase. This suggests that the electron transport rate and the quantum efficiency were not affected by water stress solely but effectively by a combination o f water stress and high temperature. The fluorescence signals related to the energy state o f the chloroplasts, i.e. the calculated energy quenching (Fig. 4b), show that the energy state o f the chloroplasts increased as a consequence o f water stress. Young leaves apparently suffered more than old leaves. The recovery after alleviation o f stress, however, was also faster in young leaves than in old leaves. The E-quenching on day 9 was not analyzed because the electron transport rate was severely impaired due to the temperature treatment. This would bias the result because the inhibited electron flow itself will lead to a slow establishment of a pH-gradient. Fig. 4c shows the exponentially fitted relation between the calculated mesophyll conductances and the energy quenching. The increased E-quenching is caused by an increase of the proton gradient due to a decrease of the ATP consumption Potato Research 32 (1989)
25
A. H. C. M. SCHAPENDONK, C. ,l. T. SPITTERS A N D P. J. GROOT Q-quenching
E-quenching
15 o
150 b
100
100
I
! 050
i
2
4
6
8
10
12
14
16
2
4
6
8
10
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14
16
Days
Daya
E-quenching
Fm/Fo
0I 07600 :x&
C
110
d
100 A
O90 LAA
\4
tx
I 020 000
L 010
I 020 Mesophy[I
i 030
= 040
conductance
D 050
070 0
t
t
t
~
h
t
2
4
6
8
10
12
i
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D~lys
Fig. 4. Time courses of fluorescence parameters for 'old' leaves (open triangles) and 'young' leaves (closed triangles), (a) Q-quenching (electron transport rate), (b) E-quenching (energy state of the leaves), (c) Relation between the mesophyll conductance (cm s ~) and the energy quenching, (d) the ratio between the maximum and the dark fluorescence (Fm/Fo) standing for membrane integrity. 26
Potato Research 32 (1989)
EFFECTS OFWATERSTRESS
in the Calvin cycle (Bradbury & Baker, 1983). The inhibited Calvin cycle apparently relates to an increase of the mesophyll conductance. However quantitative conclusions are difficult to draw because the fluorescence measurements were performed at relatively low red light intensities (100 p.mol) and thus electron transport and ATP production are expected to be rate limiting for carboxylation. The apparent accumulation of energy even under these low light conditions therefore indicates that the Calvin cycle was the main rate limiting factor at higher light intensities. The ratio F m a x / F o (Fig. 4d) is a measure of the integrity o f the system that directs the energy from the antennae pigments to photosystem II. The data indicate that the energy transfer was inhibited especially under severe drought stress (at 20 % PEG). In conclusion, water stress reduced the rate of photosynthesis at high light due to the inhibition o f the Calvin cycle. Photosynthesis at low light was probably not affected because no effect on quantum efficiency was detected.
Variations around the principal trend Consideration of the results in more detail reveals some deviations from the primary trend described above. During the first days after imposition of the stress, internal CO2 concentration was lowered to some extent and the share of the gas phase resistance was increased (Fig. 2e, f), especially in the cultivars Veenster and Alpha (Fig. 3b). On the first day, when this effect was greatest, the drop in internal CO z concentration was on the average 11%, indicating that stomatal limitation was operative shortly after exposure to water stress. Stomatal control on constant internal COz concentration was restored after a few days (Fig. 2e, 3b). The decline of stomatal limitation did increase internal CO: concentration, which effect was responsible for the slight recovery in the rate o f photosynthesis during the first days of the stress period, especially in the cultivars Veenster and Alpha (Fig. 3a). There was no active osmotic adjustment. The decline in osmotic potential, measured 7 days after onset of the stress, from - 0 . 8 0 MPa in the control to -0.83 MPa in the stress environment was fully explained by the decrease of 5 % in relative leaf-water content due to the stress. The sudden increase of temperature after day 8 resulted in a dramatic decrease of the Q-quenching, but only in the drought treatment. This reduction was completely reversed when the temperature was lowered again to 17 ~ concomitant with alleviation of the water stress (Fig. 4a). A combined water stress and heat stress seems to cause a blockage of the electron transport after photosystem II. Bilger et al. (1985) observed that heat treatment alone blocked the electron transport proportional to the measured CO2 assimilation. From the photosynthesis measurements no effects of the temperature jump would be expected (Fig. 2a). However it should be noted that these measurements were done at light saturation, where the effect of quantum efficiency is of less importance. The data in Fig. 4a suggest a reduction of the quantum efficiency by 30 % due the temperature rise from 17 ~ to 27 ~ under the stress condition.
Recovery after relief of the stress After relief of the stress, photosynthesis recovered rapidly. In old leaves, the degree of recovery was on the average 60 ~ The incomplete recovery was probably due to an acceleration of leaf senescence induced by the stress: at the end of the stress period these leaves were visually scored to be green for 62 ~ on the average, while comparable Potato Research 32 (1989)
27
A. H. C. M. SCHAPENDONK, C. J. T. SPITTERS A N D P. J. GROOT
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