Photosynthetic Acclimation in Pea and Soybean to ... - Plant Physiology

Plant Physiol. (1994) 106: 661-671

Photosynthetic Acclimation in Pea and Soybean to High Atmospheric C 0 2 Partia1 Pressure' Da-Quan Xu, Roger M. Gifford*, and W. S. Chow Commonwealth Scientific and Industrial Research Organization, Division of Plant Industry, Canberra, Australia (R.M.G., W.S.C.); and Academia Sinica, Shanghai lnstitute of Plant Physiology, Shanghai, China (D.-Q.X.)

1986), decrease in the grass Desmodium paniculatum (Wulff and Strain, 1982), and not change in water hyacinth (Larigauderie et al., 1986). There is a similar diversity of response for maximum photosynthetic capacity. Clearly, severa1 factors must be influencing these photosynthetic adjustments, such as species, plant age, leaf age, sink demand for assimilate, and the leve1 of other environmental factors, such as mineral nutrients, water supply, and temperature. The main hypotheses to explain photosynthetic adjustment in plants grown in elevated CO, are (a) feedback inhibition by accumulated carbohydrates as a result of changes in the carbohydrate source-sink interrelationships in the whole plant and (b) a physiological re-optimization of the nitrogen distribution away from the COz fixation machinery (especially RuBPCase) and toward systems to acquire other resources and establish new sinks (Bowes, 1991; Stitt, 1991). Stitt (1991) suggested that any redistribution of N in COzacclimating plants can be interpreted as a manifestation of the normal adaptive mechanisms that operate in plants to adjust photosynthetic settings to the source-sink balance of the whole plant. Our objectives were 3-fold: (a) to examine whether the amount of photosynthetic adjustment for plants grown from gennination in elevated CO, was a function of the age of the individual leaf, (b) to examine how closely changes in RuBPCase activity match leaf photosynthetic acclimation, and (c) to examine the relationship between carbohydrate accumulation and acclimation by manipulating carbohydrate sources and sinks at the same time as COz concentration.

Nonnodulated pea (Pisum safivum 1. cv Frosty) and soybean (C/ycine max [L.] Merr. cv Wye) plants were grown under artificial lights from germination with ample nutrients, 600 pmol photons m-2 s-', and either 34 t o 36 (control) or 64 t o 68 Pa (enriched) CO,. For soybean, pod removal and whole-plant shading treatments were used t o alter the source-sink balance and carbohydrate status of the plants. Crowth of both species was substantially increased by CO, enrichment despite some down-regulation of photosynthesis rate per unit leaf area ("acclimation"). Acclimation was observed in young pea leaves but not old and in old soybean leaves but not young. Acclimation was neither evident in quantum yield nor was it related t o triose phosphate limitation of net photosynthesis. A correlation between levels of starch and sugars i n the leaf and the amount of acclimation was apparent but was loose and only weakly related to the source-sink balance of the plant. A consistent feature of acclimation was reduced ribulose bisphosphate carboxylase (RuBPCase) content, although i n vivo RuBPCase activity was not necessarily diminished by elevated growth COz owing to increased percentage of activation of the enzyme. A proposal i s discussed that the complexity of photosynthetic acclimation responses t o elevated CO, i s as an expression of re-optimization of deployment of within-plant resources at three levels of competition.

Reports conceming the "acclimation" or "adjustment" of photosynthesis of leaves on plants grown continuously in elevated levels of COZare conflicting. Some indicate a substantial downward adjustment of the entire instantaneous light-saturated COz-responsecurve of leaves on plants grown at about twice normal COz levels compared with controls (e.g. in cotton [Wong, 19793, in bean [von Caemmerer and Farquhar, 19841, in water hyacinth [Spencer and Bowes, 19861, in cabbage and eggplant [Sage et al., 19891). Others report an upward adjustment of the instantaneous C0,response curve (e.g. in tomato [Hicklenton and Jolliffe, 19801, in Monterey pine [Conroy et al., 19861, in soybean [Campbell et al., 19881). Others show no difference in the response curves of control and COz-enriched plants (e.g. in wheat [Gifford, 19771, in cotton [Radin et al., 19871). Similar diversity of results exists for the initial slope of the lightresponse curve. Growth at elevated levels of C 0 2 has caused quantum yield to increase in Monterey pine (Conroy et al.,

MATERIALS A N D METHODS Plant Material

Seeds of indeterminate dwarf pea (Pisum sativum L. cv Frosty) and determinate soybean (Glycine max [L.] Merr. cv Wye) were sown in 5-L plastic pots containing a mixture of vermiculite and perlite (l:l,v/v) and grown in two cabinets (LB-type, Morse and Evans, 1962). Plants were well spaced to avoid mutual shading. In the control cabinet the partial pressure of COz was that of ambient air. In another, the partial pressure of CO, was regulated at 64 to 68 Pa (enriched) Abbreviations: P,, light- and COz-saturated leaf photosynthetic capacity; Pn, leaf net photosynthesis rate per unit area; Pn,4, Pn determined at 34 Pa COZ; RS, rookshoot dry weight ratio; RuBP, ribulose 1,5-bisphosphate; RuBPCase, ribulose 1,5-bisphosphate carboxylase.

Financia1 support for trave1 and subsistence was from the Commonwealth Scientific and Industrial Research Organization/Academia Sinica Exchange Program. * Corresponding author; fax 61-6-246-5000.

66 1

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Xu et al.

by bleeding bottled CO,, scrubbed through Purafil (Purafil, Inc., Atlanta, GA) to remove any traces of ethylene (Morison and Gifford, 1984), into the well-mixed air flow through the cabinet. COz was monitored and controlled by an IR CO, analyzer linked to a solenoid valve controller. The plants were grown under artificial light from fluorescent and tungsten filament lamps. Daylength was 12 h. Illumination was adjusted regularly to 600 pmol m-’ s-’ (400-700 nm). Air temperature was a constant 2OoC (day and night). Rhizobial inoculation was not provided. Each pot was flushed daily with full-strength Hoagland solution in the moming and watered each aftemoon. Dates of leaf emergence (defined as leaf length of 2 cm) were recorded daily to define leaf ages. Full expansion of leaves took about 7 (pea) and 14 (soybean) d. For pod remova1 experiments on soybean, a11 pods (about 60) on treated plants were removed 35 to 45 d after planting. Measurements of leaf photosynthesis and RuBPCase activity were made 10 to 14 d after pod removal, and some leaves were harvested for carbohydrate analysis. For shading experiments on soybean, whole plants were placed under shadecloth, which reduced photon flux density on upper leaves to about 90 pmol m-’s-’ for 8 d. Measurements of leaf photosynthesis and RuBPCase activity and a harvest of some leaves were made immediately before and after the shading period. Cas Exchange and Crowth Measurements

Rates of photosynthesis and transpiration per unit leaflet area were measured using a portable gas analysis system including a Parkinson leaf chamber, an IRGA, and a data logger (Analytical Development Co., Hoddesdon, Hertsfordshire, UK). Temperature, CO,, humidity, and light sensors were first calibrated. Measurements were made in the cabinets on fully expanded leaves of known ages. Irradiance at the leaf surface for a11 COz exchange measurements, except the measurement of the apparent quantum yield, was maintained at 430, 600, or 1500 pmol photons m-’ s-’. Airflow through the leaf chamber was at 0.320 L min-’. Leaf-to-air vapor pressure difference was maintained at about 1.5 kPa (pea) or 1.0 kPa (soybean). The air was obtained by mixing CO, and C02-freeair, which had been humidified by passing it through water at 19OC, using a set of Wosthoff gas-mixing pumps. Leaf temperature was sensed independently by a thermocouple touching the lower surface of the leaf. For measurement of photosynthetic CO, response, CO, partial pressure was increased stepwise from 9.5 to 95 Pa. Each COZ partial pressure was maintained for at least 10 min to reach steady-state gas exchange. For measurement of the apparent quantum yield, photon flux density was changed in eight steps from 200 to 40 pmol photon m-’ s-’. Each light intensity was maintained for at least 5 min before the measurement. For measurement of 0, sensitivity, low 0, air (2 kPa 02) was obtained by mixing pure Nz, C02, and 0 2 . Before a measurement, 0, concentration was held constant for at least 10 min. The O2sensitivity of photosynthesis was calculated as [(PnZkPa - PnZlkPa)/Pn2kPa], where Pn is the net COz fixation rate and the subscripts refer to 2 and 21 kPa 0, (Sage et al., 1989). A11 data were downloaded to a computer, and the rates of net photosynthesis (Pn), transpiration, stomatal conductance,

Plant Physiol. Vol. 106, 1994

and intercellular partial pressure of CO, were calculated using the independent leaf temperature measurement. P,,, and quantum yield were determined from the steadystate rate of 0, evolution by leaf discs in whitl. light from a slide projector using a Hansatech (King’s Lynn, UK) leaf disc O2electrode. Measurements were made at a leaf temperature of 25OC in saturating COz conditions (1 kPa C 0 2 from a 1 M carbonate/bicarbonate buffer solution at pH 9). Quantum yield was determined as the 0, evolved per absorbed photon over the range of irradiance where light Wa:j the limiting factor Photosynthetic capacity was measured as the lightand C0,-saturated rate of O, evolution per unit leaf area. Plants were harvested after 5 to 9 weeks of growth. Total leaf area per plant was measured with an electronic area meter (model LT-3000; Li-Cor, Lincoln, NE). Roots, stems, and leaves were dried to a constant weight at 130°C. RuBPCase Activity and Activation Assay

Leaf discs were cut, wrapped in aluminum foil, immediately frozen, and stored in liquid N2. Measurements of RuBPCase activity were based on the method of von Caemmerer and Edmondson (1986). A leaf disc (3.4 cm’) ivas ground in liquid NZ.After the N, had evaporated, 1 mL of COn-free0.1 M Bicine (pH 8.1) was added. The assay was then conducted either immediately or after the leaf extract had been held in the assay buffer at 25OC for 7 min. For the assay, 25 pL of leaf extract were added to 420 pL of the assay buffer including 0.1 M Bicine-KOH with 20 mM MgC1’ (pH 8.10), 20 pL of 0.2 M NaH14C03(final concentration 15 m, 10 Bq nmol-’), and 10 NL of 2.5 m 6-phosphogluconate. The reaction was started within 10 s using 25 pL of RuBP (20 I ~ M stock) and stopped, after incubation at 25OC for 60 s, wiíh 100 p L of 2 M formic acid and dried on a hot plate. Then, 3130 pL of water and 2.7 mL of scintillation fluid were added. For blanks no RuBP was added, but the acid was used. For standards RuBP was not added. Assay mixture (20 pL) was piit into 480 pL of buffer. After the sample was mixed, 50 pL were removed and nixed with 50 p L of 0.1 M NaOH (to keep alkaline), and 200 pL of water and 2.7 mL of scintillation fluid were added. Photosynthate and Chl Analysis

Leaf samples were frozen in liquid N2 antl freeze-dried. Starch, SUC,Glc, and Fru were determined by proprietary enzymatic methods based on the determination of Glc-6-P (Boehringer Mannheim GmbH, Mannheim, Germany; D-G~c/ UV methodkit 716-251). Chl was determinedin 80% acetone with a Hitachi U-3200 spectrophotometer (Por:a et al., 1989). RESULTS

Severa1 experiments, involving measurenients through time and comparing pea and soybean responses to elevated COz,were conducted on intact plants grown without shading and with pods retained (TablesI-V; Fig. 1).To:al dry weights of the spaced plants were increased by 27 to ,48% (pea) and 37 to 57% (soybean) by elevated COz (Table I). These increases were partially attributable to increased total leaf area, owing to the formation of more leaves at elevated CO, in

663

Photosynthetic Acclimation to C 0 2 both species. Specific leaf weight was little affected by COZ doubling for pea but was increased in soybean. R:S ratio was increased substantially in soybean and in one pea experiment (Table I). In both species, for 11- to 14-d-old leaves, growth at elevated C 0 2 caused a decrease of about 30% in stomatal conductance and an increase of 25% in Pn compared with the controls grown in normal air when measured at their growth CO, partial pressures and irradiances (Table 11). The increase in Pn also partially accounted for the increased dry weight of plants grown at elevated CO,. When measured at saturating irradiance, the net photosynthetic response to intercellular C 0 2 partial pressure changed with leaf age for leaves of both species. For pea (Fig. la), both the initial slope of the light-saturated CO,-response curve and photosynthetic rates at high C 0 2 were slightly decreased by CO, doubling during growth in younger leaves (leaf age 9-13 d) but not in older leaves (leaf age 22-25 d). By contrast, for soybean (Fig. lb), C 0 2 doubling caused a large difference in the response curve in older soybean leaves (leaf age 21-26 d) but no difference in the younger leaves. Thus, considerable downward adjustment of soybean photosynthesis occurred after the soybean leaf was fully expanded. Apparent quantum yield of photosynthesis measured on intact plants at 34 Pa CO, was not significantly affected by

high CO, partial pressure during growth for either pea or soybean. The apparent quantum requirements were 24.1 f 0.7 and 23.0 f 0.2 for young pea leaves grown at 34 and 68 Pa, respectively, and 30.5 k 1.7 and 34.2 +- 1.7 for old soybean leaves. Quantum yield based on the Oz yield per photon absorbed in the leaf disc 0, electrode under saturating CO, was not affected either (see discussion in Tables V, VII, and XII). O2 sensitivity of net photosynthesis (Table 111) was determined for those leaf ages that showed the most downregulation under high CO, (i.e. young leaves in pea and old leaves in soybean, Fig. 1). The high O2 sensitivity of Pn and its independence of growth CO, (Table 111) indicates that there was no tiose phosphate limitation to Pn in the downregulated leaves (see later). Photorespiration rates, expressed as the difference of Pns measured at 2 and 21 kPa Oz, were little affected in either species by the growth CO, doubling when measured at the same CO, partial pressure of 34 Pa. Carbohydrate contents of leaves collected in the early afternoon, including Glc, Fru, SUC,and starch, were increased substantially in both pea and soybean as a result of growth at elevated CO, concentration. For both species these increases were relatively larger in the young leaves than in the older leaves (Table IV). RuBPCase activity, Chl, and P,,, were determined on leaves of each species at the ages that gave maximal down-

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Table 1. Effects of COzenrichment (68 Pa) on growth of pea (cv Frosty) and soybean (cv Wye) For each species two data sets from different experiments are presented for plants harvested at different times after sowing. SED is the standard error of the difference between means; n is the

number of redicates. Per Plant GrowthC02 Concentration

Age at Harvest

Pa

d

Leaf area

No. of

leaves

Total dry wt

g

cm2

Specific Leaf Wt

R:S

mg cm-'

Pea: Experiment 1 34 68 SED % lncrease

34 68

5 5

42 42

OO /

YO lncrease

2.7 2.9 0.01 8

0.21 0.20 0.014 -4

2.6 2.6 0.1 3 2

0.32 0.44 0.052 40

2.4 2.7 0.09

Pea: Experiment 2 16.5 261 5 3447 24.4 325 1.3 32 48

6 6

34 34

Soybean: Experiment 1 1832 9.07 2371 14.20 135 0.35 29 57

13

0.26 0.31 0.02 16

Soybean: Experiment 1930 90 2208 99 254 8.7 14 10

7.2 10.1 0.5 41

0.27 0.32 0.015 19

lncrease

34 68 SED

14.0 17.8 1.9 27

55 55

lncrease

34 68 SED

62 72 3.7 16

6 6

SED ? O '

2255 2610 355 15

6 6

64 64

2 44.54 61.10 7.96 37

Xu et al.

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Effects of C 0 2 doubling on leaf gas exchange The measurements were made on leaves at their growth C 0 2 partial pressure, 21 kPa O,, and 430 pmol photons m-, s-'. Leaf ages were 11 to 14 d and 12 d, respectively, for pea and soybean; n = 9 or 10. Results are means with SE in parentheses. a and mean, respectively, P < 0.05 and P < 0.01 for differences between plants grown in control air and enriched CO,. Ci, CO, partial pressure of intercellular space, Pa; C,, stomatal conductance, mo1 HZO m-' s-'; Pn, pmol CO, m-' s-'; J , transpiration rate, mmol HzO m-'. s-'; T/Pn, transpiration ratio, mo1 HzO/mol C02;JL,leaf temperature. "C: EIC refers to the ratio of enriched C0,:control data. Table II.

Species

Pea

Crowth

C,

coz Control Enriched E/C

Soybean

Control Enriched E/C

C,

Pn

T/Pn

1

TL

27.5 (0.2) 55.7 (0.3) 2.0

0.69 (0.05) 0.4gb (0.03) 0.70

12.5 (0.3) 15.7b (0.4) 1.25

4.5 (0.07) 4.2" (0.1) 0.93

363 (9) 271 (8) 0.75

21.1 (O. 1) 21 .8b (0.1)

28.0 (0.2) 56.6 (0.3) 2.02

0.60 (0.02) 0.44b (0.01) 0.72

12.9 (0.2) 16.0b (0.3) 1.25

4.1 (0.05) 3.8b (0.1) 0.93

320 (7) 238b (4) 0.75

21.9 (0.04) 22.6b (0.03)

regulation at high COz. COz doubling caused a significant decrease in RuBPCase activity at its in vivo level of activation in young pea leaves but not in the older soybean leaves (Table V). The maximally activated activity was decreased in both species when grown at elevated C 0 2 . Thus, the percentage of activation in vivo was increased for soybean at high COz, but the level of activation was unaffected in pea. CO, doubling had little effect on Chl per unit leaf area of either species, and thus the maximally activated activity of RuBPCase per unit Chl tended to decrease (Table V). High COz partial pressure during growth had no significant effect on P,,, as measured by O2exchange under saturating light and C 0 2 for either species (Table V). C02-saturatedquantum yields were little affected by growth CO,.

The experiments in which sources and sinks were manipulated by pod remova1 (Tables VI-VIU) and shading (Tables IX-XII) were conducted only on soybean. Removing the filling soybean seed pods caused a significant reduction of Pn and stomatal conductance of the older leaves when measured at 34 Pa CO, (Table VI). This effect w3s of similar magnitude to the effect of growing soybean plants under elevated COz. These two effects were also synergistic for both Pn and stomatal conductance (Table VI). Similarly, pod remova1 reduced in vivo RuBPCase activity by about the same amount as did growing the plants at elevatecl CO, (Table VII). Chl density (m-'), however, showed a different response: pod remova1 had no effect on Chl m-' in control COz plants, but doubling the C 0 2 concentration rediiced Chl m-'

Effects of O, partial pressure on Pn The measurements were made at 1500 pmol photons m-'s-', 34 Pa CO,, and 22°C. Leaf ages were 13 to 14 d and 23 to 27 d, respectively, for pea (five samples) and soybean (eight samples). Results are means with SE in parentheses. "',a, and mean, respectively, P > 0.05, P < 0.05, and P < 0.01 for differences between plants grown in control air and enriched CO,. E/C refers to the ratio of enriched C02:controldata.

Table 111.

Crowth Species

coz

Net Photosynthesis 21 kPa02

2 kPa O,

Photorespiration

o 2

Sensitivity

+mo/ CO, m-2 s-'

Pea

Control Enriched E/C

Soybean

Control Enriched E/C

18.8 (0.7) 17.4"' (0.2) 0.92

25.0 (1.O) 23.9"' (0.6) 0.96

6.2 (0.4) 6.6"' (0.4) 1.O6

24.7% (1.O%) 2 7.2%"' (1.O%) 1.10

13.5 (0.7) 9.8b (0.7) 0.72

17.2 (0.7) 13.0b (1.O) 0.75

3.7 (0.3) 3.2"' (0.4) 0.86

21.8% (1.7%) 24.3%"' ( 1.3%) 1.12

Photosynthetic Acclimation to CO,

665

Table IV. Effects of C 0 2 doubling on photosynthate contents of leaves with different leaf age in pea and soybean grown at 20°C

E/C refers to the ratio of enriched C0,:control data. Species

Leaf Age

Gowth

d

Pea

Clc

Fru

Starch

mg dm-’

mg dm-*

mg dm-’

co,

10-14

Control Enriched

22-26

Control Enriched E/C

7-14

Control Enriched

E/C

Soybean

E/C

22

Control Enriched

33-36

Control Enriched

E/C

EIC

especially in the absence of pods (Table VII). Whereas elevated growth C 0 2 tended to increase the percentage of activation of RuBPCase (Tables V and VII), pod removal had no effect (Table VII). P,,, was much more strongly diminished by de-podding than by growth under elevated COz, and these two effects were synergistic (Table VII), as they were for Pn. Pod remova1 caused a much bigger increase in starch accumulation in the leaf than &d growing the plants under elevated CO,, and the two effects combined were a little stronger than additive (Table VIII). Shading soybean plants for 8 d was effective at reducing nonstructural carbohydrate levels, especially starch and Suc levels, in the leaves to very low levels, which did not differ between C 0 2 treatments (Table IX). The effect of shading on the hexose levels was less clear than it was for starch. Despite the massive decrease in starch levels, shading did not cause

SUC mg dm-’

11.1 19.1 1.72 10.6 12.6 1.19

20.2 33.1 1.64 22.5 24.8 1.10

18.6 36.0 1.93 8.9 9.8 1.11

9.9 14.9 1.51 11.5 13.9 1.21 17.3 24.1 1.39

4.6 9.9 2.13 5.8 6.3 1.O8 11.0 18.5 1.68

59.5 145.1 2.44 113.6 186.8 1.65 402.6 739.8 1.84

23.9 39.1 1.64 26.5 27.8 1.O5

Pn3* of high C02-grownplants to recover fully to the rate for shaded, ambient C02-grown plants (Tables X and XI), although there was partial recovery. P,,, of high COz-grown plants did recover fully to the shaded, ambient CO, values (Table XII), even though the fully activated RuBPCase leve1 after shading was greatly diminished by high CO, during growth. The RuBPCase information is incomplete for the shading experiment, so interpretation in terms of percentage of activation is not possible (Table XII). DISCUSSION

The photosynthetic acclimation responses to COz described are complex, partially inconsistent, and difficult to understand. As an aid to interpretation, we focus discussion by examining the compatibility of the results with the hypothesis Figure 1. C 0 2 response of net photosynthesis at different leaf ages of plants grown at control and elevated C 0 2 levels. Young leaves, open symbols: 9- to 13-d-old for pea and 12- to 14-

d-old for soybean. Old leaves, filled symbols: 22- to 25-d-old for pea and 21- to 26-d-old for soybean. Control growth CO, data are shown as circles. Enriched growth-C02data are shown as triangles. The measurements were made at 1500 pmol photons m-’ s-’ and 21 to 22°C. n = 8 or 10. The numbers show the decrease in Pn caused by CO, doubling during growth expressed in percentages of the control. ns, Means P > 0.05; *, P < 0.05; *, P < 0.01.

20 40 60 20 40 60 lntercellular space CO, partial pressure (Pa)

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Plant Physiol. Vcd. 106, 1994

Table V. €ffects of C 0 2 doubling on Chl content, RuBPCase activity, P,,,

and quantum yield at saturating C 0 2 in pea and soybean Results are means with SE in parentheses ( n = 3 or 4). "', a, and mean, respectively, P > 0.05, P < 0.05, and P < 0.01 for differences between plants grown in control air and enriched. E/C refers to the ratio of enriched COz:control data. Leaf ages were about 10 and 20 d, respectively, for pea and soybean. Measurements of ,,P , at 2000 pmol photons m-' s-' and quantum yield were made using a leaf disc oxygen electrode at 25°C and saturated C 0 2 . Parameter Chl (pmol m-') RuBPCase activity (pmol C 0 2 m-2 s-') In vivo Maximum RuBPCase,,/C hl (mo1 C 0 2 mol-' Chl s-l) ?40 Activation (in vivo/maximum) P,, (pmol Ozm-2s-1) (mmol O2 mol-' Chl

Crowth CO, (pea)

-

Crowth COz (soybean)

Control

Enriched

E/C

Control

Enriched

E/C

491 (13)

474"' (20)

0.97

736 (30)

661"' (22)

0.90

65.8 (2.4) 81.5 (4.7) 0.17 (0.01)

44.8b (1.8) 55.Sb(2.8) 0.12b (0.01)

0.68 0.68 0.70

41.2 (0.8) 89.3 (4.9) 0.12 (0.01)

44.0"' (1.5) 1.07 70.0a(1.8) 0.78 0.1 1"' (0.00) 0.87

80.0 (3.0)

81.0"' (3.0)

1.01

46.0 (3.0)

63.0b (2.0)

1.39

48.0 (0.7) 115 (8.3)

50.2"' (2.8) 117"' (4.3)

1.05 1.O2

37.8 (2.3) 55.4 (3.9)

41.2"' (0.8) 57.3"' (0.6)

1.09 1.O3

S-1)

Quantum yield (mo1 Oz/mol absorbed photons)

0.100 (0.003)

0.097"' (0.004) 0.97

that photosynthetic adjustment to elevated COz is an expression of normal metabolic adjustments that have evolved in plants to match supply and demand for carbohydrate and to optimize the deployment of resources such as nitrogen. Key results pertinent to the discussion are: 1. Continuously elevated CO, concentrations increased the photosynthesis rate (Table 11) and carbohydrate content (especially starch) of leaves (Table IV) and the whole-plant dry matter growth rate (Table I) of pea and soybean plants. This was despite some down-regulation of Pn in plants from elevated CO, conditions when measured at ambient CO, levels in saturating light (Pn34)(Fig. 1). 2. There was no effect of growth C 0 2 level on quantum yields of either species (Tables V, VII, and XII). 3. Down-regulation of Pn34was observed only for young leaves of (indeterminate) pea plants (Fig. la) and for old leaves of (determinate) soybean plants (Fig. lb). 4. Both young pea leaves and old soybean leaves that showed down-regulation of photosynthesis when grown at high C 0 2 concentration exhibited strong O2dependence of Pn34(Table 111). 5. The down-regulation of Pn34in young pea leaves at high growth C 0 2 level was weak (Fig. la) but was accompanied by a strong diminution of RuBPCase activity, with no change in the percentage of activation level of RuBPCase (Table V) but relatively large increases in leaf starch and sugar levels (Table IV). The down-regulation of Pn34in old soybean leaves at high growth C 0 2 level was strong but accompanied by little or no reduction in RuBPCase activity because the reduced fully activated RuBPCase activity was offset by increased percentage of activation level of RuBPCase (Table V) and by relatively less increase in starch and sugar levels in

0.100 (0.004) 0.089"' (0.012) 0.89

the leaves than was observed for the younger leaves that did not acclimate (Table IV). 6. Where down-regulation due to elevated CIO2 occurred, it was associated with an increase in nonstructural carbohydrate levels, particularly starch, in the leaf, but the converse was not true: it was possible to have large increases of leaf starch and sugars without any photosynthetic ilown-regulation (Table IV cf. Fig. l b for young soybean leates). Also, Pn of soybean in control C 0 2 was unaffected by age (Fig. 1) despite a large increase in starch with age (Table IV). Contrary to this, Pn of pea decreased markedly with age while starch level declined (Fig. 1; Table IV). 7. Complete pod removal in soybean had an effect similar to elevated COz on Pn34and on nonstructural carbohydrate buildup in older soybean leaves (Tables VI and VIII). 8. Combined pod remova1 and elevated gro,vth C 0 2 had a synergistic effect on Pn34 but an approximately additive effect on starch levels of soybean (Tables VI and VIII). 9. Drastically reducing leaf nonstructural carbohydrate levels by shading for several days did not fully restore down-regulated Pn34 of elevated-C02 plants to the level of the ambient C02, but shaded, controls (Tables X and XI). One approach to understanding carbohydrale source-sink relationships in plants is to focus on the tissiie content of mobilizable carbohydrates themselves as medi,itors that can directly or indirectly drive sink growth or inhibit photosynthesis by some variant of end product inhibition. According to Stitt (1991), accumulation of sugars and starch in leaves might act directly or indirectly to inhibit p hotosynthesis through several routes involving different time scales: 1. The idea that large starch granules physically disrupt chloroplast function (Cave et al., 1981), althsugh not yet

667

Photosynthetic Acclimation to COz

Table VI. Effects of pod removal on photosynthetic acclimation to CO, doubling in soybean leaves Results are means with SE in parentheses. "',', and mean, respectively, P > 0.05, P < 0.05, and P < 0.01 for differences between plants grown in control air and enriched COz. E/C refers to the ratio of enriched C0,:control data. Leaf ages were 24 to 29 d old. Measurements were made at 600 pmol photons m-2 s-', a leaf temperature of 26 to 27"C, and 34 Pa COz 12 d after removal of all pods. R/W refers to the ratio of data for pods removed:pods retained. Ci, COzpartia1 pressure of intercellular space, Pa; C,, stomatal conductance, mo1 H 2 0 m-'s-'; Pn, pmol C 0 2 m-'s-'. Crowth Condition Treatment

with pods

Parameter

Ci

h4

Pod removal

EIC

Control

Enriched

19.4 (0.2) 0.1 3 (0.02) 10.3 (0.5) 20.2 (0.8) 0.10 (0.01) 7.7 (1.0) 103 77 74

21 .4b (0.4) 0.10" (0.01) 7.4b (0.6) 22.0"' (1.4) 0.04b (0.01) 2.5b (0.8) 105 37 34

disproven, seems unlikely to be operative as a sensitive feedback mechanism, except perhaps insensitively at the extreme. 2. Direct feedback regulation of photosynthesis via the inhibition of Suc synthesis from triose phosphate precursors exported from the chloroplast to the cytosol, could lead to Pi shortage in the chloroplast for photosynthesis and to decreased triose phosphate export. This would cause starch to accumulate in the chloroplast. Although such a mechanism might operate during short-tem transients of source-sink mismatch, it is unlikely to be a long-tenn mechanism of feedback regulation because it would not optimize the use of the leaf's metabolic resources. A test for this kind of feedback

1.10 0.79 0.72 1.12 0.39 0.33

involving failure to recycle Pi back from the chloroplast is the 0, sensitivity of net photosynthesis (Sharkey, 1985): 0,insensitive net photosynthesis is Pi limited. In the present study net photosynthesis of both pea and soybean, whether from elevated COnor not, was highly O2sensitive when the measured leaves were at the ages when they showed photosynthetic down-regulation (Table 111) and contained high levels of starch (Table IV). Therefore, this b e c t mechanism of feedback down-regulation can be discounted in this study. 3. Indirect regulation of photosynthesis could involve decreased levels of photosynthetic enzymes, especially RuBPCase. This might come about in two ways. First, the photosynthetic system might "recognize" that at elevated CO,

Table VII. Effects of pod removal on Chl content, RuBPCase activity, photosynthetic capacity, and quantum yield in the soybeans leaves Results are means with SE in parentheses (n = 3 or 4). "',a, and mean, respectively, P > 0.05, P < 0.05, and P < 0.01 for differences between plants grown in control air and enriched CO,. E/C refers to the ratio of enriched COz:control data. Leaf ages were 24 to 29 d old. Measurements of P,, at 2000 pmol photons m-' s-' and quantum yield were made using a leaf disc 0, electrode at 25°C and saturated COz 12 d after removal of all pods. Crowth CO, (with pods)

Crowth CO, (pods removed)

Parameter

Chl (pmol m-Z)

RuBPCase activity (pmol COz rn-, s-') In vivo Maximum Ru B PCase,,JC hl O/O

(mo1 CO, mol-' Chl s-') Activation (in vivo/maximum)

Control

Enriched

E/C

Control

Enriched

E/C

699 (36)

586" (17)

0.83

732 (22)

453b (45)

0.62

51.3 (3.0) 39.6a (2.9) 0.77 41.9 (1.6) 28.5b (1.0) 0.68 68.4 (6.4) 43.6" (2.2) 0.64 51.9 (5.5) 31.8" (0.4) 0.61 0.097 (0.005) 0.074a (0.005) 0.75 0.071 (0.010) 0.072"' (0.007) 0.99

76 (5)

92"'(11)

1.21

83 (12)

90"'(2)

1.08

p,, (pmol O2m-'s-') (mmol O2mol-' Chl s-')

Quantum yield (mo1 02/mol

photons)

42.3 (1.2) 37.3" (1.5) 0.88 29.6 (1.2) 18.0b (1.5) 0.61 64.1 (4.2) 62.5"'(2.2) 0.98 42.4 (1.6) 47.1"' (7.3) 1.11 0.097 (0.007) 0.092"' (0.004) 0.95 0.089 (0.003) 0.085"' (0.004) 0.96

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Effects of pod removal on photosynthate contents in the soybean leaves Results are means with SE in parentheses for six replicates. Leaves were harvested 15 d after pod removal from three replicate plants. Leaf ages were 26 to 3 d old. ns, a, and mean, respectively, P > 0.05, P < 0.05, and P < 0.01 for differences between plants grown in control air and enriched C02. Glc is free Glc, Fru is total Fru moiety free and combined, and SUCis based o n its Glc content. E/C refers to the ratio of enriched C07:control data. Table VIII.

’

Photosynthate Conients (mg dm-’ leaf area)

Crowth

Treatment

coz Control

with pods

Enriched E/C

Pods removed

Control Enriched E/C

Clc

Fru

5.4 (0.9) 7.6“ (1.5) 1.40 9.4 (2.3) 11.5”’ (1.O) 1.23

5.1 (0.4) 7.8” (1.3) 1.53 11.2 (2.1) 12.3”’ (1.O) 1.10

suc

Starch

3.2 (0.6) 5.2” (1.1) 1.61 6.9 (1.94) 6.5”’ (1.3) 0.95

162 (21) 363’ (24) 2.20 458 (32) 701’ (36) 1.50

sinks and/or into the systems for acquiring other resources such as roots for mineral nutrient acquisition. This is suggested by the observation (Conroy, 1992) that plants from high CO, conditions always have a lower foliar Iq concentration than control plants, even when grown with abundant N supply. This was also true when foliar N concentration was expressed on a structural (rather than total) leaf dry weight basis (Rogers et al., 1993). For both pea and soybean the increased branching (Table I) was an increased investment in sinks, as was the increase in pod numbers (data not recorded). In addition, increased R:S ratio (dry weight bmis) at high growth CO, may represent a redistribution oi investment from leaf function to root function (Table I), biit we do not know whether the R:S ratio on an N basis followed that pattem. That the investment in aspects of both the light and dark reactions of photosynthesis was reduced (see above) also suggests that this second route of resource n2deployment was followed by plants in this study. Where altemative sinks were not possible, because pods had been removed from plants that had already ceased new leaf production and probably new root formation too, both RuBPC’ase and Chl levels decreased equally such that the RuBPC ase:Chl ratio was unaffected by growth C 0 2 (Table VII). However, where heavy short-term shading of pod-filling plants decreased the source:sink ratio, the elevated growth CO, concentration did

there is a need for a different balance of investment in the COZ fixation versus light reaction machinery of photosynthesis and redistibute N from the former to the latter. Review of the evidence (Makino, 1994) has indicated that the proportional investment of leaf N in RuBPCase and electron transport capacity did not change with the CO, concentration during growth. For example, in five species the ratio of RuBPCase:Chl (an indicator of N investment in the dark reactions relative to light reactions [Evans and Terashima, 19871) was not affected by growth in elevated CO, (Sage et al., 1989). However, in the present study, although the RuBPCase:Chl ratio did decrease under elevated CO, by 30% in pea and by 13 to 25% in soybean with pods still attached (Tables V and VII), the changes in ratio were not because Chl levels increased but because fully activated RuBPCase levels decreased considerably. In fact, Chl levels tended to decrease too. It seems then that the relative N investment in both the dark and light reactions per unit leaf area probably declined under elevated CO,. Thus, although the plant may have changed the distribution of N between the COZfixation and light-harvesting/electron transport sides of photosynthesis, a second effect may be present too. The second potential indirect response to growth under high CO, concentration is a change in the relative distribution of N between the photosynthetic machinery as a whole and

Effects of whole-plant shading on photosynthate contents in soybean leaves SE in parentheses for three replicate experiments. None of the E/C differences are significant (P > 0.05). Leaves were harvested 2 d before and 8 d after shading three replicate plants. Leaf ages were 24 to 29 d old and 34 to 37 d old, respectively, for before and after shading. Here, Glc is free Clc, Fru is total Fru moiety free and combined, and Suc is based o n its Clc content. E/C refers to t h e ratio of enriched C02:control data. Table IX.

Values are means with

Shading Treatment

Before

coz Control Enriched

E/C After

Photosynthate Contents (mg dm-2 leaf area)

Crowth

Control

Enriched E/C

Clc

Fru

7.2 (1.3) 9.6 (2.2) 1.34 7.1 (1.1) 9.5 (1.4) 1.35

6.5 (0.8) 9.6 (2.9) 1.48 3.0 (0.2) 4.0 (0.5) 1.33

SUC

3.7 (1.8) 7.0 (3.1) 1.89 1.3 (0.5) 1.3 (0.5) 0.97

Starch

135 (19.8) 246 (87) 13 2 20 (6.4) 22.1 (7.0) 1.O8

669

Photosynthetic Acclimation to CO,

Table X. Effectsof whole-plant shading on photosynthetic acclimation to C02 doubling in the soybean leaves

Results are means with SE in parentheses. "',a, and mean, respectively, P > 0.05, P < 0.05, and P < 0.01 for differences between plants grown in control air and enriched C 0 2 . C,, C 0 2 partial pressure of intercellular space, Pa; G,, stomatal conductance, mo1 H 2 0 m-'s-'; PnJ4,pmol COZm-2 S-'. E/C refers to the ratio of enriched C02:controldata. n = 10. Leaf ages were the same as those in Table IX. Measurements were made at 600 umol Dhotons m-2 s-' and a leaf temDerature of 27°C. Crowth C 0 2

Shading Treatment

Before shading

Parameter

C,

G 8 d after shading

Ph4 C,

G, Pr-13~

Control

Enriched

E/C

20.3 (0.4) 0.19 (0.01) 13.0 (0.4) 20.4 (0.3) 0.18 (0.01) 1 1 .O (0.5)

20.0"' (0.3) O.llb(O.O1) 8.3b(0.4) 20.5"'(0.4) 0.14"(0.01) 9.0"(0.5)

0.98 0.55 0.64 1 .o1 0.77 0.82

lead to much lowered RuBPCase:Chl ratio (Table XII), presumably as N was shifted to light-harvesting machinery. Another aspect of photosynthetic regulation is the degree of activation of RuBPCase. This is under complex dynamic metabolic control in relation to the environment, especially irradiance. The intracellular leve1 of CO, is also involved in RuBPCase activation through its reaction with the enzyme Rubisco activase (Lan and Mott, 1991). In this study, as in others (Tissue et al., 1993, for loblolly pine), RuBPCase activation in intact soybean plants was increased by elevated C 0 2 from 46 to 63% (Table V) or from 76 to 92% (Table VII). In pea, however, the percentage of activation of RuBPCase remained at 80% with or without the elevated CO,. These results contrast with those of Sage et al. (1989) in which percentage of activation decreased with increased growth C 0 2 in Chenopodium album, Phaseolus vulgaris, Brassica oleracea, Solanum tuberosum, and Solanum melongena. Such longterm deactivation was seen as a failure of the plants to acclimate optimally to the elevated growth CO,, N being wasted in unutilized RuBPCase (as indicated by its fully activated activity). This was not the case here but quite the

converse for soybean, in which strong down-regulation of net photosynthesis in the older leaves (Fig. lb) and buildup of leaf starch was accompanied by a decreased amount of RuBPCase operating at a higher percentage of activation at high COz, thereby allowing a given amount of N to serve a larger leaf area. However, other observations on soybean (Campbell et al., 1988) did not show increased RuBPCase activation in elevated CO, or decreased RuBPCase activity. Indeed, Campbell et al. found up-regulation rather than down-regulation of photosynthesis. Thus, there appear to be severa1 possible adjustment responses in leaf photosynthesis to continuous growth in elevated CO,, depending on conditions yet to be clearly defined. In soybean substantial down-regulation occurred only in the older leaves (Fig. lb). These measurements were made at a time when the terminal apex was floral and thus further vegetative growth had ceased. The dominant sinks were the existing pods; few new sinks were possible. Under the conditions involved, leaf starch built up rapidly in older control leaves and elevated CO, caused an increase in starch and sugars to very high levels (Table IV). By contrast, in pea,

Table XI. Effects of whole-plant shading on photosynthetic acclimation to enriched COz in the soybean leaves measured at control and enriched CO, partial pressure Results are means with SE in parentheses for 10 leaves. "',', and mean, respectively, P > 0.05, P < 0.05, and P < 0.01 for differences between plants grown in control air and enriched COz. E/C refers to t h e ratio of enriched C02:control data. Leaf ages were 29 to 34 and 42 to 47 d old, respectively, for before and after shading plant. Measurements were made at 600 pmol photons m-2 s-', a leaf temperature of 27"C, and control and enriched C 0 2 partial pressure. C,, C 0 2 partial pressure of intercellular space, Pa; Cs, stomatal conductance, mo1 H2O m-2 s-'; Pn, pmol COz m-2 S-'.

Shading Treatment

Control Measurement C 0 2

Enriched Measurement C 0 2

Crowth C 0 2

Growth CO,

Parameter Control

Before shading

Ci

G, Pn

8 d after shading

C,

G, Pn

Enriched

19.5 (0.3) 20.0"' (0.2) 0.22 (0.01) 0.094b(0.005) 12.7 (0.3) 6.1b(0.3) 22.2 (0.3) 20.6b(0.4) 0.15 (0.01) 0.076b(0.003) 8.3 (0.8) 5.5b(0.2)

E/C

1 .O3 0.42 0.48 0.93 0.52 0.66

Control

Enriched

E/C

41.5 (0.3) 37.6b(0.7) 0.91 0.22 (0.01) 0.094b(0.004) 0.42 20.9 (0.6) 12.6b(0.5) 0.60 49.4 (0.8) 40.8b(0.6) 0.83 0.19 (0.02) 0.07gb(0.002) 0.41 12.1 (1.0) lO.O"'(O.4) 0.83

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Plant Physiol. Vol. 106, 1994

Table XII. Effects of shading plant on Chl content, RuBPCase activity, P,,,,,, and quaritum yield in the soybean leaves Results are means with SE in parentheses for three or four leaves. "',', and rnean, respectively, P > 0.05, P < 0.05, and P < 0.01 for differences between plants grown in control air and enriched C 0 2 . E/C refers to the ratio of enriched C02:control data. Leaf ages were the same as those in Table IX. Conditions of measurements were the same as those in Table VI1 for P,, and auantum vield. Parameter

Chl (w"/m2)

Crowth CO, (before shading)

Crowth CO, (after shading)

-

Control

Enriched

E/C

Control

Enriched

659 (19)

477" (37)

0.72

690 (26)

555"' (75)

0.80

63.4 (4.3) 0.092

30.5b (1.7) 0.055

0.48 0.60

26.5 (2.7) 42.9 (1.9) 0.090 (0.006)

27.3"' (3.6) 55.4"' (4.9) 0.088"' (0.002)

1 .O3 1.29 0.98

RuBPCase activity (pmol COz m-'s-') In vivo maximum Ru BPCase,,,/Chl (mo1 C 0 2 mol-' Chl s-')

E/C

Pmax

(pmol O2 m-'s-')

(mmol O2 mol-' Chl s-') Quantum yield (mo1 02/mol photons)

44.4 (2.6) 67.3 (1.9) 0.099 (0.003)

29.3a (3.7) 60.9"' (0.3) 0.097"' (0.001)

which was generating new pods continuously, the older leaves lost starch, relative to the younger, and CO, did not increase the starch level. These leaves did not down-regulate photosynthesis at high CO,. On the other hand, the young pea leaves, having modest starch and sugar levels, did down-regulate under elevated CO,. These leaf age and species comparisons are partially supportive of the concept that sink potential acting via mechanisms involving carbohydrate buildup in the leaves were causing the down-regulation of photosynthesis. The pod removal and shading experiments were designed for soybean to probe whether the carbohydrate buildup can account entirely for the observed down-regulation. The results suggest that sink potential and associated carbohydrate levels did not account fully for the acclimation observed on intact plants, although there seems to be a substantial involvement as discussed below. The diminution of Pn34 of old soybean leaves caused by growing plants under elevated CO, was similar in magnitude to the diminution of Pn34 caused by complete pod removal (Table VI) as was the decline in RuBPCase activity in vivo (Table VII). By contrast, however, growth at elevated CO, caused a much smaller increase in leaf starch content than did pod removal (Table VIII). Thus, there was some additional source of photosynthetic down-regulation in the high COZgrown material that was not attributable solely to the nonstructural carbohydrate buildup arising from change in sourcesink ratio. In addition, there was a synergistic effect on Pn34 of both growing the plants at elevated COZ and removing pods (Table VI), whereas the starch increase in the leaves was close to being additive (Table VIII). This again suggests that there was something more to photosynthetic down-regulation under elevated COz than could be related linearly to leaf starch levels. The shading experiments supported this suggestion.The old leaves on soybean plants from normal air had lower Pn34values after 8 d or more of shading

0.66 0.90 0.98

(Tables X and XI). At the same time their starch contents declined to very low levels, which were uninfluenced by the CO, concentration during growth (Table IX). Yet the Pn3* of the plants grown under elevated CO, level before and during the shading was not restored to the Pn34of control CO, plants following shading (Tables X and XI), i.e. there, was some down-regulation that was unrelated to the starch level. Thus, photosynthetic down-regulation in these experiments occurred at specific leaf ages that differed for the two species. Where it occurred it was broadly but not precisely correlated with conditions under which sink growth was likely to be unable to respond to a higher photosynthetic input, such that starch levels built up in the leaves. The metabolic basis of down-regulation was related to reduced RuBPCase activity owing to reduced fully acivated RuBPCase activity (i.e. reduced RuBPCase amount), which was often partially compensated by increased levels 01 percentage of RuBPCase activation. It was neither related to triose phosphate feedback limitation nor to photosynthetic quantum efficiency. However, there was not a tight link between leaf starch or sugar levels and the amount of down-regulation, suggesting that leaves that are acclimating in aesponse to source-sink imbalance induced by elevated CO: conditions are not doing so in wholly the same manner as those that are acclimating to source-sink imbalance brought abciut by other environmental circumstances. Our results support the idea that deployment o f resources, especially N, in the photosynthetic system, particularly RuBPCase, is central to COz acclimation induced by any circumstance. They also show that photosynthetic downregulation from elevated COz has similarities with, but is not identical with, down-regulation following pod re moval. There appear to be three levels of resource competition operating, each potentially related to N deploynient within the plant, that determine the degree of photosynthetic acclimation to elevated CO,. The first is the partiticning of re-

Photosynthetic Acclimation to C 0 2

sources within the leaf between photosynthetic carboxylation machinery and light-harvesting/electron transport machinery, i.e. higher CO, concentration tends to foster lower carboxylation capacity (e.g. lower RuBPCase:Chl ratio; Tables V, VII, and XII) if plant growth is substantially source limited but not when it is strongly sink limited after pod remova1 (Table VII). The second is the deployment of resources between the photosynthetic machinery as a whole and the carbohydrate utilization machinery (i.e. sourcesink ratio). Higher growth CO, concentration tends to lead to less investment in overall photosynthetic machinery in favor of more sinks if growth is strongly sink limited. The third leve1 of competition,which may influence whether photosynthesis acclimates to elevated CO,, relates to the relative deployment of plant resources for carbon acquisition from the atmosphere (and accumulation of the products in sinks) versus the acquisition of soil resources (water and nutrients). Shortage of water or nutrients would make investment in roots particularly critica1 to plant growth. Elevated CO, offers the opportunity for investment in root without loss of capability for carbon acquisition and utilization (i.e. increased R:S, see Table I). Whether increased R:S involves parallel change in partitioning of N remains to be established. It appears that a11 three tiers of resource competition leading to photosynthetic acclimation to CO, were observed in the present study, sometimes concurrently. This may explain the lack of a clear-cut explanation of the cause of downregulation. But the three-tier concept should form a framework for designing further experiments to discriminate between the various acclimation-inducing circumstances to provide a basis for modeling photosynthetic acclimation to elevated CO,. ACKNOWLEDCMENTS

We thank the staff of the phytotron in Division of Plant Industry, Commonwealth Scientific and Industry Research Organization, for help with the experiments. We also appreciate the help of Mr. Martyn England and Ms. Stefanie Wojtkewicz during the study. Received March 17, 1994;accepted June 22, 1994. Copyright Clearance Center: 0032-0889/94/106/066l/l1. LITERATURE ClTED

Bowes G (1991)Growth at elevated COz: photosynthetic responses mediated through Rubisco. Plant Cell Environ 1 4 795-806 Campbell WJ, Allen LH Jr, Bowes G (1988)Effects of COz concentration on Rubisco activity, amount, and photosynthesis in soybean leaves. Plants Physiol88: 1310-1316 Cave G, Tolley LC, Strain BR (1981) Effect of carbon dioxide enrichment on chlorophyll content, starch content and starch grain structure in Trifolium subtevaneum leaves. Physiol Plant 51: 171-174 Conroy JP (1992)Influence of elevated atmospheric COz concentration on plant nutrition. Aust J Bot 4 0 445-456

671

Conroy JP, Smillie RM, Kuppers M, Bevege DI, Barlow EW (1986) Chlorophyll a fluorescence and photosynthetic and growth responses of Pinus radiata to phosphorus deficiency, drought stress, and high C02. Plant Physiol81: 423-429 Evans JR, Terashima I (1987) Effects of nitrogen nutrition on electron transport components and photosynthesis in spinach. Aust J Plant Physioll4 59-68 Gifford RM (1977)Growth pattem, carbon dioxide exchange and dry weight distribution in wheat growing under differing photosynthetic environments. Aust J Plant Physiol4 99-110 Hicklenton PR, Jolliffe PA (1980)Alterations in the physiology of C 0 2 exchange in tomato plants grown in C 0 2 enriched atmospheres. Can J Bot 5 8 2181-2189 Lan Y, Mott KA (1991)Determination of apparent K, values for ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase using the spectrophotometric assay of Rubisco activity. Plant Physiol95 604-609 Larigauderie A, Roy J, Berger A (1986)Long term effects of high CO, concentration on photosynthesis of water hyacinth (Eichhornia crassipea (Mart.) Solms).J Exp Bot 37: 1303-1312 Makino A (1994)Biochemistry of C3-photosynthesisin high CO2. J Plant Res 107: 79-84 Morison JIL, Gifford RM (1984)Plant growth and water use with limited water supply in high CO, concentrations. I. Leaf area, water use and transpiration. Aust J Plant Physiol 11: 361-374 Morse RN, Evans LT (1962)Design and development of CERESan Australian phytotron. J Agric Eng Res 7: 128-140 Porra RJ, Thompson WA, Kriedemann PE (1989)Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophyll a and b with four different solvents: verifications of the concentrations of chlorophyll standards by atomic absorbtion spectroscopy. Biochim Biophy Acta 975 384-394 Radin JW, Kimball BA, Hendrbt DL, Mauney JR (1987)Photosynthesis of cotton plants exposed to elevated levels of carbon dioxide in the field. Photosynth Res 1 2 191-203 Rogers GS, Payne L, Milham P, Conroy J (1993)Nitrogen and phosphorus requirements of cotton and wheat under changing atmospheric COz concentrations. Plant Soil155/156: 231-234 Sage RF, Sharkey TD, Seemann JR (1989)Acclimation of photosynthesis to elevated COz in five CB speaes. Plant Physiol 8 9 590-596 Sharkey TD (1985)O,-insensitive photosynthesis in C3 plants. Its occurrence and a possible explanation. Plant Physiol78 71-75 Spencer W, Bowes G (1986)Photosynthesis and growth of water hyacinth under CO, enrichment. Plant Physiol82 528-533 Stitt M (1991)Rising COz levels and their potential sigruficance for carbon flow in plant cells. Plant Cell Environ 1 4 741-762 Tissue DT, Thomas RB, Strain BR (1993)Long term effects of elevated CO, and nutrients on photosynthesis and Rubisco in loblolly pine seedlings. Plant Cell Environ 1 6 859-865 von Caemmerer S, Edmondson DL (1986)Relationship between steady-state gas exchange, in vivo ribulose bisphosphate carboxylase activity and some carbon reduction cycle intermediates in Raphanus sativus. Aust J Plant Physioll3 669-688 von Caemmerer S, Farquhar GD (1984)Effects of partial defoliation, changes of irradiance during growth, short-term water stress and growth at enhanced p (C02) on the photosynthetic capacity of leaves of Phaseolus vulgaris L. Planta 160 320-329 Wong SC (1979)Elevated atmospheric partial pressure of CO2 and plant growth. I. Interactions of nitrogen nutrition and photosynthetic capacity in C3 and C, plants. Oecologia 44:68-74 Wulff RD, Strain BR (1982)Effect of COz enrichment on growth and photosynthesis in Desmodium paniculatum. Can J Bot 60: 1084-1091