Planta
Planta (Berl.) 132, 1 9 - 2 3 (1976)
9 by Springer-Verlag 1976
Carbon Dioxide Exchange in Relation to Sink Demand in Wheat H.M. Rawson, R.M. Gifford, and P.M. Bremner Division of Plant Industry, CSIRO, P.O. Box 1600, Canberra City, A.C.T. 2601, Australia
Summary. In this paper, experiments are described which examine the effect of requirement for assimilates by the ear on the rate of net photosynthesis in leaves of wheat (Triticum aestivum L.). Different levels of requirement were achieved by various levels of sterilization of florets just before anthesis, which resulted in a range of grain numbers per ear, and by inhibiting photosynthesis of the intact ear by 3(3,4-dichlorophenyl)- 1,1-dimethylurea (DCMU). Only the ear and two uppermost leaves of the main shoot were considered, all the lower leaves and tiller leaves being excised when the experimental treatments were imposed. In two experiments, tiller regrowth was permitted during the experimental period, while in a third, new tillers were defoliated regularly. The response of leaf photosynthesis to the level of assimilate requirement by the ear was influenced by the treatment of the vegetative tillers. Thus, the net photosynthesis rate of the flag leaf was decreased by a reduction in grain number, or increased by inhibition of photosynthesis in the ear, only when the vegetative tillers were kept defoliated; when these tillers were allowed to re-grow normally, there was no influence of ear treatment on leaf photosynthesis. Temporal changes in leaf photosynthesis were consistent with this response pattern, i.e., when tillers were defoliated, the initial high rates of photosynthesis persisted for much longer. In the experiment where photosynthesis was influenced by the requirement for assimilate in the ear, the variation occurred through change in stomatal conductance on the abaxial surface of the leaf. This surface has a lesser conductance to CO2 exchange than the adaxial surface. The implication of this finding to rapid methods of plant screening is discussed.
Introduction
Birecka and Dakic-Wlodkowska (1963), King et al. (1967) and others observed a strong dependence of the rate of photosynthesis in the flag leaf of wheat on the level of requirement for assimilate by the developing grains. Such a dependence was also indicated by work on a range of wheat cultivars showing that the net CO2 exchange (NCE) rate of the flag leaf commonly declined just after anthesis, rising again when rapid grain-filling started (Yordanov and Belikov, 1962; Birecka and Dakic-Wlodkowska, 1963; Evans and Rawson, 1970; Rawson and Evans, 1971). This apparent dependence of the leaf NCE rate on the level of demand for assimilates by the ear (which we will call ~ dependent response") has been widely quoted as implying that the assimilatory capacity in wheat may exceed the capacity of the grains to store assimilate. Studies by Lupton (1968), Apel etal. (1973) and Austin and Edrich (1975), however, led them to conclude that photosynthesis was independent of the level of assimilate requirement by the ear ("independent response"). In this paper we show that both "dependent" and "independent" responses of flag leaf photosynthesis can be found within the same experimental material, depending on the way the tillers of the plants are managed experimentally. Materials and Methods Cultural Conditions The seeds (Triticum aestivum L. cv. Kalyansona, an Indian cultivar derived from Mexican semi-dwarf stock and provided by the Indian Agricultural Research Institute, New Delhi) were grown in 15 x 8cm pots containing a mixture of perlite and vermiculite, at a stand density of 130 plants m-2. Water and a Hoagland No. 2 nutrient solution were provided daily. Tillers were cut back to just above ground level 4 times weekly. Experiments 1 and 3 were
20
H.M. Rawson et al.: C O 2 Exchange and Sink D e m a n d in Wheat
carried out in parallel on the same batch of plants raised in summer in a glasshouse of the Canberra phytotron (21 ~ for 8 h and 16 ~ for the remainder of the day; photoperiod was extended to 16 h with low intensity light from incandescent lamps). Plants for Experiment 2 were raised in a u t u m n in a similar glasshouse set at 15/10 ~ and a photoperiod of 16 h. After ear emergence the plants were transferred to cabinets providing 830 gE m - 2 s-1 of photosynthetically active radiation, from Philips Cool-white fluorescent lamps, at ear height. For Experiments 1 and 3, the cabinets were set at 20 ~ for an 18 h day and 10 ~ for the dark period. In Experiment 2 daily assimilation was reduced by providing only 9 h illumination in the 20/10 ~ cabinet, the photoperiod being extended to 18 h by low-intensity light from incandescent lamps. Treatments Plants with 22 spikelets on the ear of the main shoot were selected after ear emergence. A n y tiller regrowth was removed and all leaf laminae but the uppermost two were cut off from the main shoots. In Experiment 1, treatments were imposed 1 or 2 days before anthesis and were: (1) control--ear left intact (78 grains); (2)--as in (1) except that the ears were dipped for 20 s in 5 x 10 - s D C M U (3-(3,4-dichlorophenyl)-l, 1-dimethylurea) to inhibit their photosynthesis (a concomitant effect was a 10% reduction in grain set); (3), (4) and ( 5 ) - s e l e c t e d florets were sterilized so that either 30, 40 or 60 grains remained in the ear. The florets were sterilized from apex to base of each spikelet. The procedure worked well (see Table 1). In Experiment 1 the tillers were allowed to regrow from the day treatments were imposed. In Experiment 3 the treatments corresponded to treatments (1), (2) and (3) above except that trimming of the tillers down to ground level was repeated regularly, without however removing their apices. The D C M U treatment of the ear was repeated twice when the measurements of photosynthesis, routinely done in Experiment 1, showed that photosynthesis was recovering. Experiment 2 consisted of treatments (1) and (3) above and a treatment involving shading (50% light interception) of the ear. Also, the daily radiation integral on the plants was reduced in this experiment (see above).
such that CO2 was never depleted more than 40 g] 1- i. Two replicates, each of either 4 leaves or 4 ears, were measured twice weekly in each experiment. The plants were harvested immediately after measurement. Gas analysis of the flag leaves in Experiment 3 was performed with an apparatus designed for simultaneous measurements of CO2 and water vapour exchange on each surface of an 8 x 1 cm segment in the central portion of the attached leaf, using an open system of air flow (Gifford and Marshall, 1973). Carbon dioxide concentration was measured with a G r u b b Parson's SB2 infrared gas analyser and water vapour with a dewpoint hygrometer. Carbon dioxide in the leaf chamber was controlled close to 295 gl lon each side of the leaf, relative humidity at 46 %, and leaf temperature at 24.5 ~ The light source, placed about 30 cm above the chamber, was a mercury vapour lamp which delivered 1,200 gE m - 2 s 1 of photosynthetically active radiation at the upper (adaxial) leaf surface. Air recirculation by centrifugal blowers built into the chamber reduced boundary-layer resistance to CO 2 diffusion on each surface to 0.73 s c m - 1 . For each leaf, on each occasion, the CO 2 compensation point was determined by measuring CO2 flux at a concentration very close to the compensation point and extrapolating linearly to the axis for zero net CO2 exchange. Four flag leaves were measured in each treatment at intervals of from 3 to 5 days for a total of 39 days, the same leaves being used throughout. Calculation of the gas phase conductances and sub-stomatal C O : concentration was performed for each leaf surface separately. A single internal or residual conductance was calculated for each leaf by regarding the mean calculated sub-stomatal CO 2 concentration (averaged across the two leaf surfaces) and the CO 2 compensation point as determining the CO 2 gradient, and the total leaf N C E as the flux (see Jarvis, 1971, for the method of calculating gas-phase and residual conductances). In Experiment 1 the dry weights of grain, chaff, stem, leaves, tillers and roots were recorded for six plants of each treatment twice weekly, and a final harvest of 11 plants was made at maturity, 5 weeks after anthesis. In Experiments 2 and 3, only a final harvest was made.
Results
Measuremen ts
Experiments 1 and 2," Normal Tiller Regrowth
In Experiments 1 and 2 gas exchange was measured under the light and temperature conditions of the cabinet in which the experiments were conducted. The measurements were made with a Hartm a n n and Braun U R A S II infrared gas analyser. Flow rates were
The treatments imposed in experiment 1 gave a more than 2 fold range in grain weight per ear at maturity (Table 1). As the treatments did not affect the dura-
Table 1. Grain n u m b e r s and weights in Expts. 1 a n d 2 •
Treatment
standard error of the mean
Grain weight per ear (mg)
Grain no. per ear
Weight per grain (mg)
Experiment 1 Control DCMU 60 grains 40 grains 30 grains
3,428 _+ 142 3,017• 36 2,678 +_ t 02 1,914 • 96 1,397_+ 95
78 • 3 71• 60 _+2 41 • 2 30•
43.6 _+0.4 42.4• 44.7 • 0.5 46.3 +_0.5 45.9_+0.8
Experiment 2 Control Ears shaded 30 grains
2,993 _+ 128 2,825 • 74 1,664• 55
66 • 2 69 _+2 27_+ I
45.2 • 0.6 40.1 _+0.5 60.6•
H.M. Rawson et al.: CO2 Exchange and Sink Demand in Wheat
mental treatments on dry weight at maturity were negligible. The only significant difference between Experients 1 and 2 was the reduction in plant dry weight at maturity to 12.20 g in the latter experiment. The inference from both experiments was that the plant responded to a change in the requirement of the ear for assimilates by altering the distribution of assimilate rather than by changing photosynthetic rate. In a third experiment, however, we obtained different results.
70-
"g--...6
50
rq"-.O []
tO r-
21
30
c7 *~ l0
O
Experiment 3; Tillers Defoliated Regularly
Z
I
I
I
10 20 30 Days from anthesis
I
Z,0
Fig. 1. The time course of net C O 2 exchange for flag and penultimate leaves in Expt. 1. Values for only the extreme treatments are shown, [] DCMU on the ears; o Control; zx 30 grains per ear. Solid lines are for the flag leaf, broken lines for the penultimate leaf
tion of grain growth, this range was caused by differences in the grain-growth rates which were proportional to the number of grains present. Grain growth rates were extremely fast, being 140 mg d a y - ~ e a r in the control plants between 20 and 30 days after anthesis. Despite the large differences in the requirements of grain growth for assimilates, net photosynthesis of neither the flag leaf not the penultimate leaf differed between the treatments at any stage (Fig. 1). N o r had these treatments any effect on the N C E of the ear; mean rates of ear photosynthesis _+ the standard error of the mean for the period of rapid grain-filling were 702+_115, 810+_126, 756+_ 129, 799+_ 115, and - 4 1 8 + 147 ng COa s ~ for the control, 60 grains, 40 grains, 30 grains and D C M U ear treatments, respectively. The dry weights of the plants at maturity were consistent with this pattern; the only significant difference (p < 0.05) was a reduction from 15.88 to 14.29 g per plant caused by the application of D C M U to the ears. Inhibition of ear photosynthesis, which in other treatments averaged 600 ng CO2 s- ~ e a r - t over the grain filling period, would account for much of this reduction. Under the conditions of Experiment 1 there appeared, then, to be no feedback effect of ear requirement for assimilates on leaf photosynthesis. This lack of effect could have been because assimilates were non-limiting as a consequence of the long (18 h) daily photosynthetic period during grain filling. However, although we halved this period in Experiment 2, the results (not presented) were essentially the same: the patterns and rates of photosynthesis were similar to those of Figure 1, and the effects of the various experi-
Experiment 3 was run concurrently with Experiment 1, using plants selected from the same batch and grown during the experimental period in the same cabinet. Their treatment differed in one r e s p e c t - i n Experiment 3 the regrowing tillers were defoliated regularly to just above ground level, but without removing the tiller apices, the cut leaf sheaths always being visibly elongated on the day following defoliation. Both the time course of phosynthesis and the results of the treatments of the ear differed from those of Experiment 1. First, to demonstrate the overall effects of tiller defoliation, the results of all grainremoval treatments were averaged and plotted against time (Fig. 2). Leaf N C E did not begin to decline until 3 weeks after anthesis. Indeed, there was some rallying 90 T u)
80
moE70
~ ~ i / ~ ~
NCEtot
\;
Planta (Berl.) 132, 1 9 - 2 3 (1976)
9 by Springer-Verlag 1976
Carbon Dioxide Exchange in Relation to Sink Demand in Wheat H.M. Rawson, R.M. Gifford, and P.M. Bremner Division of Plant Industry, CSIRO, P.O. Box 1600, Canberra City, A.C.T. 2601, Australia
Summary. In this paper, experiments are described which examine the effect of requirement for assimilates by the ear on the rate of net photosynthesis in leaves of wheat (Triticum aestivum L.). Different levels of requirement were achieved by various levels of sterilization of florets just before anthesis, which resulted in a range of grain numbers per ear, and by inhibiting photosynthesis of the intact ear by 3(3,4-dichlorophenyl)- 1,1-dimethylurea (DCMU). Only the ear and two uppermost leaves of the main shoot were considered, all the lower leaves and tiller leaves being excised when the experimental treatments were imposed. In two experiments, tiller regrowth was permitted during the experimental period, while in a third, new tillers were defoliated regularly. The response of leaf photosynthesis to the level of assimilate requirement by the ear was influenced by the treatment of the vegetative tillers. Thus, the net photosynthesis rate of the flag leaf was decreased by a reduction in grain number, or increased by inhibition of photosynthesis in the ear, only when the vegetative tillers were kept defoliated; when these tillers were allowed to re-grow normally, there was no influence of ear treatment on leaf photosynthesis. Temporal changes in leaf photosynthesis were consistent with this response pattern, i.e., when tillers were defoliated, the initial high rates of photosynthesis persisted for much longer. In the experiment where photosynthesis was influenced by the requirement for assimilate in the ear, the variation occurred through change in stomatal conductance on the abaxial surface of the leaf. This surface has a lesser conductance to CO2 exchange than the adaxial surface. The implication of this finding to rapid methods of plant screening is discussed.
Introduction
Birecka and Dakic-Wlodkowska (1963), King et al. (1967) and others observed a strong dependence of the rate of photosynthesis in the flag leaf of wheat on the level of requirement for assimilate by the developing grains. Such a dependence was also indicated by work on a range of wheat cultivars showing that the net CO2 exchange (NCE) rate of the flag leaf commonly declined just after anthesis, rising again when rapid grain-filling started (Yordanov and Belikov, 1962; Birecka and Dakic-Wlodkowska, 1963; Evans and Rawson, 1970; Rawson and Evans, 1971). This apparent dependence of the leaf NCE rate on the level of demand for assimilates by the ear (which we will call ~ dependent response") has been widely quoted as implying that the assimilatory capacity in wheat may exceed the capacity of the grains to store assimilate. Studies by Lupton (1968), Apel etal. (1973) and Austin and Edrich (1975), however, led them to conclude that photosynthesis was independent of the level of assimilate requirement by the ear ("independent response"). In this paper we show that both "dependent" and "independent" responses of flag leaf photosynthesis can be found within the same experimental material, depending on the way the tillers of the plants are managed experimentally. Materials and Methods Cultural Conditions The seeds (Triticum aestivum L. cv. Kalyansona, an Indian cultivar derived from Mexican semi-dwarf stock and provided by the Indian Agricultural Research Institute, New Delhi) were grown in 15 x 8cm pots containing a mixture of perlite and vermiculite, at a stand density of 130 plants m-2. Water and a Hoagland No. 2 nutrient solution were provided daily. Tillers were cut back to just above ground level 4 times weekly. Experiments 1 and 3 were
20
H.M. Rawson et al.: C O 2 Exchange and Sink D e m a n d in Wheat
carried out in parallel on the same batch of plants raised in summer in a glasshouse of the Canberra phytotron (21 ~ for 8 h and 16 ~ for the remainder of the day; photoperiod was extended to 16 h with low intensity light from incandescent lamps). Plants for Experiment 2 were raised in a u t u m n in a similar glasshouse set at 15/10 ~ and a photoperiod of 16 h. After ear emergence the plants were transferred to cabinets providing 830 gE m - 2 s-1 of photosynthetically active radiation, from Philips Cool-white fluorescent lamps, at ear height. For Experiments 1 and 3, the cabinets were set at 20 ~ for an 18 h day and 10 ~ for the dark period. In Experiment 2 daily assimilation was reduced by providing only 9 h illumination in the 20/10 ~ cabinet, the photoperiod being extended to 18 h by low-intensity light from incandescent lamps. Treatments Plants with 22 spikelets on the ear of the main shoot were selected after ear emergence. A n y tiller regrowth was removed and all leaf laminae but the uppermost two were cut off from the main shoots. In Experiment 1, treatments were imposed 1 or 2 days before anthesis and were: (1) control--ear left intact (78 grains); (2)--as in (1) except that the ears were dipped for 20 s in 5 x 10 - s D C M U (3-(3,4-dichlorophenyl)-l, 1-dimethylurea) to inhibit their photosynthesis (a concomitant effect was a 10% reduction in grain set); (3), (4) and ( 5 ) - s e l e c t e d florets were sterilized so that either 30, 40 or 60 grains remained in the ear. The florets were sterilized from apex to base of each spikelet. The procedure worked well (see Table 1). In Experiment 1 the tillers were allowed to regrow from the day treatments were imposed. In Experiment 3 the treatments corresponded to treatments (1), (2) and (3) above except that trimming of the tillers down to ground level was repeated regularly, without however removing their apices. The D C M U treatment of the ear was repeated twice when the measurements of photosynthesis, routinely done in Experiment 1, showed that photosynthesis was recovering. Experiment 2 consisted of treatments (1) and (3) above and a treatment involving shading (50% light interception) of the ear. Also, the daily radiation integral on the plants was reduced in this experiment (see above).
such that CO2 was never depleted more than 40 g] 1- i. Two replicates, each of either 4 leaves or 4 ears, were measured twice weekly in each experiment. The plants were harvested immediately after measurement. Gas analysis of the flag leaves in Experiment 3 was performed with an apparatus designed for simultaneous measurements of CO2 and water vapour exchange on each surface of an 8 x 1 cm segment in the central portion of the attached leaf, using an open system of air flow (Gifford and Marshall, 1973). Carbon dioxide concentration was measured with a G r u b b Parson's SB2 infrared gas analyser and water vapour with a dewpoint hygrometer. Carbon dioxide in the leaf chamber was controlled close to 295 gl lon each side of the leaf, relative humidity at 46 %, and leaf temperature at 24.5 ~ The light source, placed about 30 cm above the chamber, was a mercury vapour lamp which delivered 1,200 gE m - 2 s 1 of photosynthetically active radiation at the upper (adaxial) leaf surface. Air recirculation by centrifugal blowers built into the chamber reduced boundary-layer resistance to CO 2 diffusion on each surface to 0.73 s c m - 1 . For each leaf, on each occasion, the CO 2 compensation point was determined by measuring CO2 flux at a concentration very close to the compensation point and extrapolating linearly to the axis for zero net CO2 exchange. Four flag leaves were measured in each treatment at intervals of from 3 to 5 days for a total of 39 days, the same leaves being used throughout. Calculation of the gas phase conductances and sub-stomatal C O : concentration was performed for each leaf surface separately. A single internal or residual conductance was calculated for each leaf by regarding the mean calculated sub-stomatal CO 2 concentration (averaged across the two leaf surfaces) and the CO 2 compensation point as determining the CO 2 gradient, and the total leaf N C E as the flux (see Jarvis, 1971, for the method of calculating gas-phase and residual conductances). In Experiment 1 the dry weights of grain, chaff, stem, leaves, tillers and roots were recorded for six plants of each treatment twice weekly, and a final harvest of 11 plants was made at maturity, 5 weeks after anthesis. In Experiments 2 and 3, only a final harvest was made.
Results
Measuremen ts
Experiments 1 and 2," Normal Tiller Regrowth
In Experiments 1 and 2 gas exchange was measured under the light and temperature conditions of the cabinet in which the experiments were conducted. The measurements were made with a Hartm a n n and Braun U R A S II infrared gas analyser. Flow rates were
The treatments imposed in experiment 1 gave a more than 2 fold range in grain weight per ear at maturity (Table 1). As the treatments did not affect the dura-
Table 1. Grain n u m b e r s and weights in Expts. 1 a n d 2 •
Treatment
standard error of the mean
Grain weight per ear (mg)
Grain no. per ear
Weight per grain (mg)
Experiment 1 Control DCMU 60 grains 40 grains 30 grains
3,428 _+ 142 3,017• 36 2,678 +_ t 02 1,914 • 96 1,397_+ 95
78 • 3 71• 60 _+2 41 • 2 30•
43.6 _+0.4 42.4• 44.7 • 0.5 46.3 +_0.5 45.9_+0.8
Experiment 2 Control Ears shaded 30 grains
2,993 _+ 128 2,825 • 74 1,664• 55
66 • 2 69 _+2 27_+ I
45.2 • 0.6 40.1 _+0.5 60.6•
H.M. Rawson et al.: CO2 Exchange and Sink Demand in Wheat
mental treatments on dry weight at maturity were negligible. The only significant difference between Experients 1 and 2 was the reduction in plant dry weight at maturity to 12.20 g in the latter experiment. The inference from both experiments was that the plant responded to a change in the requirement of the ear for assimilates by altering the distribution of assimilate rather than by changing photosynthetic rate. In a third experiment, however, we obtained different results.
70-
"g--...6
50
rq"-.O []
tO r-
21
30
c7 *~ l0
O
Experiment 3; Tillers Defoliated Regularly
Z
I
I
I
10 20 30 Days from anthesis
I
Z,0
Fig. 1. The time course of net C O 2 exchange for flag and penultimate leaves in Expt. 1. Values for only the extreme treatments are shown, [] DCMU on the ears; o Control; zx 30 grains per ear. Solid lines are for the flag leaf, broken lines for the penultimate leaf
tion of grain growth, this range was caused by differences in the grain-growth rates which were proportional to the number of grains present. Grain growth rates were extremely fast, being 140 mg d a y - ~ e a r in the control plants between 20 and 30 days after anthesis. Despite the large differences in the requirements of grain growth for assimilates, net photosynthesis of neither the flag leaf not the penultimate leaf differed between the treatments at any stage (Fig. 1). N o r had these treatments any effect on the N C E of the ear; mean rates of ear photosynthesis _+ the standard error of the mean for the period of rapid grain-filling were 702+_115, 810+_126, 756+_ 129, 799+_ 115, and - 4 1 8 + 147 ng COa s ~ for the control, 60 grains, 40 grains, 30 grains and D C M U ear treatments, respectively. The dry weights of the plants at maturity were consistent with this pattern; the only significant difference (p < 0.05) was a reduction from 15.88 to 14.29 g per plant caused by the application of D C M U to the ears. Inhibition of ear photosynthesis, which in other treatments averaged 600 ng CO2 s- ~ e a r - t over the grain filling period, would account for much of this reduction. Under the conditions of Experiment 1 there appeared, then, to be no feedback effect of ear requirement for assimilates on leaf photosynthesis. This lack of effect could have been because assimilates were non-limiting as a consequence of the long (18 h) daily photosynthetic period during grain filling. However, although we halved this period in Experiment 2, the results (not presented) were essentially the same: the patterns and rates of photosynthesis were similar to those of Figure 1, and the effects of the various experi-
Experiment 3 was run concurrently with Experiment 1, using plants selected from the same batch and grown during the experimental period in the same cabinet. Their treatment differed in one r e s p e c t - i n Experiment 3 the regrowing tillers were defoliated regularly to just above ground level, but without removing the tiller apices, the cut leaf sheaths always being visibly elongated on the day following defoliation. Both the time course of phosynthesis and the results of the treatments of the ear differed from those of Experiment 1. First, to demonstrate the overall effects of tiller defoliation, the results of all grainremoval treatments were averaged and plotted against time (Fig. 2). Leaf N C E did not begin to decline until 3 weeks after anthesis. Indeed, there was some rallying 90 T u)
80
moE70
~ ~ i / ~ ~
NCEtot
\;
