Leaf Photosynthesis and Respiration of High CO2-Grown ... - NCBI

Plant Physiol. (1992) 98, 949-954

Received for publication June 6, 1991 Accepted August 20, 1991

0032-0889/92/98/0949/06/$01 .00/0

Leaf Photosynthesis and Respiration of High CO2-Grown Tobacco Plants Selected for Survival under CO2 Compensation Point Conditions1 Esteban Delgado, Joaquim Azcon-Bieto*, Xavier Aranda, Javier Palazon, and Hipolito Medrano Laboratori Fisiologia Vegetal, Departament de Biologia Ambiental, Institut d'Estudis AvanQats-Universitat de les Illes Balears, 07071 Palma de Mallorca, Spain (E.D., H. M.); Unitat de Fisiologia Vegetal, Departament de Biologia Vegeta/, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain (J.A.-B., X.A.); and Unitat de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, 08028 Barcelona, Spain (J. P.) ABSTRACT

Screening procedures based on survival under CO2 compensation point conditions for obtaining lines of C3 plants with higher photosynthesis and/or lower photorespiration have not been very successful (10, 21, 24, 26), even though the simplicity and possibility of large-scale application ofthese screening methods fueled the hopes of overcoming the main technical limitations of selection methods based on direct measurements of photosynthesis rate, as was pointed out by Nasyrov (22). Medrano and Primo-Millo (19) developed a screening chamber in which haploid tobacco (cv Wisconsin-38) plants were selected by survival after a 45-d period under 60 ppm CO2 in the circulating air. Haploids were obtained from ethylmethylsulfonate-treated anthers. These selected haploid plants showed a higher growth rate, greater leaf area, and plant production under greenhouse conditions. The floral buds of surviving haploids were treated with colchicine, and diploid fertile flowers were developed. After self-pollination, viable seeds were obtained from five selected lines. A series of field assays showed consistent advantage of selected SP lines2 in plant dry matter production in comparison with the unselected source cultivar (20). These field assays also showed a consistent increase in plant leaf area in those lines. Nevertheless, CO2 assimilation rate of detached leaves did not show substantial differences among selected lines and the control one (12, 13). Recent results suggest that the specificity factor of Rubisco did not change in these lines in response to the selection procedure (E. Delgado, M. Parry, D.W. Lawlor, A.J. Keys, and H. Medrano, unpublished results). In the present paper, we report studies of the photosynthetic and respiratory characteristics of the low CO2 survival-selected tobacco lines when grown at high CO2 levels. The growth responses of the selected lines to high CO2 were better than those of the parental line, and this is remarkable because these plants were initially selected for survival under low CO2. We have confirmed the absence of important differences in photosynthesis rates per unit leaf area and CO2 compensation point, but significant reduction of mature leaf respiration per unit dry weight was observed in selected lines which could

Four self-pollinated, doubled-haploid tobacco, (Nicotiana tabacum L.) lines (SP422, SP432, SP435, and SP451), selected as haploids by survival in a low CO2 atmosphere, and the parental cv Wisconsin-38 were grown from seed in a growth room kept at high CO2 levels (600-700 parts per million). The selected plants were much larger (especially SP422, SP432, and SP451) than Wisconsin-38 nine weeks after planting. The specific leaf dry weight and the carbon (but not nitrogen and sulfur) content per unit area were also higher in the selected plants. However, the chlorophyll, carotenoid, and alkaloid contents and the chlorophyll a/b ratio varied little. The net CO2 assimilation rate per unit area measured in the growth room at high CO2 was not higher in the selected plants. The CO2 assimilation rate versus intercellular CO2 curve and the CO2 compensation point showed no substantial differences among the different lines, even though these plants were selected for survival under CO2 compensation point conditions. Adult leaf respiration rates were similar when expressed per unit area but were lower in the selected lines when expressed per unit dry weight. Leaf respiration rates were negatively correlated with specific leaf dry weight and with the carbon content per unit area and were positively correlated with nitrogen and sulfur content of the dry matter. The altemative pathway was not involved in respiration in the dark in these leaves. The better carbon economy of tobacco lines selected for low CO2 survival was not apparently related to an improvement of photosynthesis rate but could be related, at least partially, to a significantly reduced respiration (mainly cytochrome pathway) rate per unit carbon.

The relationship between leaf photosynthesis rate and plant productivity is difficult to establish even though dry matter accumulation obviously reflects the efficiency of the plant photosynthetic processes (14, 23). It is well accepted that total canopy photosynthesis during the growth period is closely related to yield, as has been reported in several species (4, 29). However, many attempts to improve leaf photosynthesis per unit area by genetic selection did not result in any substantial increase of crop productivity (23, 24, 27).

'Supported by the Programa Nacional de Investigaci6n Agricola, PLANICYT, Spanish Government, grant No. AGR89-580.

2

Abbreviations: SP lines, self-pollinated lines; CCCP, carbonylcyanide m-chlorophenyl hydrazone. 949

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help to explain, at least in part, the higher growth performance of these plants. MATERIALS AND METHODS Plant Material

Self-pollinated, doubled-haploid tobacco (Nicotiana tabacum) lines (SP422, SP432, SP435, and SP45 1), selected as haploids by survival in a low CO2 atmosphere (near the CO2 compensation point [19]), and the parental cv Wisconsin-38 were grown from seed in a growth room kept at high CO2 levels (600-700 ppm). The substrate used was vermiculite. Plants were watered every day, and nutrients were given two or three times a week with full-strength Hewitt solution (7). Photosynthetic quantum flux density was about 300 ,umol. m2s , and the day/night temperature regimen was 24/ 2 1C, with a daylength of 13 h. RH during the day was between 35 and 50%. Plants used in the experiments were 11 to 12 weeks old after planting, still in the vegetative state. Analysis of Leaf Components Specific leaf dry weight was determined by drying the leaves in an oven at 70 to 80C until their weights were constant. Carbon, nitrogen, and sulfur contents were determined from dry biomass with a Carlo Erba NA 1500 elemental analyzer by the staff of the Serveis Cientifico-T&cnics of the University of Barcelona. Chl and carotenoid contents were determined spectrophotometrically from leaf discs according to the method of Lichtenthaler and Wellburn (17). The content of alkaloids (mainly nicotine) was determined from dried leaves according to the method described by Saunders and Blume (25). Leaves were ground and 500 mg of dry powder was extracted with 10 mL of 25 mm sodium phosphate buffer (pH 7.8) at room temperature for 24 h under constant agitation. The aqueous extract was ultracentrifuged and filtered through a 0.45-tim Millipore filter. The samples were stored in sealed vials at -20°C. The HPLC system used consisted of an LKB 2158 Uvicord SD detector provided with a 254-nm filter, 2150 HPLC pump, and 2210 recorder. Nicotine was quantitatively separated using a Waters u-Bondapak C18 reversephase column (30- x 0.4-cm), eluted with an isocratic mobile phase of 40% (v/v) methanol containing 2% (v/v) phosphoric acid buffered to pH 7.25 with triethylamine, at a flow rate of 0.8 mL/min. The peak areas corresponding to nicotine of the samples (20,uL injection volume), which had the same retention time of authentic nicotine (Merck), were integrated by comparison with an external standard calibration curve (r = 0.995). Stomatal density and index were counted from several randomly chosen fields (400-fold magnification) on epidermal strips obtained from both the adaxial and abaxial tobacco leaf surfaces. Gas Exchange Measurements Net CO2 exchange rates were measured on attached fully expanded leaves using a portable gas exchange system (Li-Cor LI-6200 portable photosynthesis system, Lincoln, NE). Net CO2 assimilation rate was determined in situ in the growth room at the C02, light, temperature, and humidity growth

Plant Physiol. Vol. 98, 1992

conditions (see "Plant Material" above). Different external CO2 concentrations around the enclosed leaf were generated by mixing the air of the growth room with C02-free air. The gas exchange system included correction for leaks at the lower CO2 concentrations to avoid underestimation of the assimilation rate and, thus, overestimation ofthe CO2 compensation point (18). Measurements were made during the first hours of the day period. The rate of respiratory CO2 release in the dark of attached adult leaves was measured at 2 1°C at the end of the night period, using the same Li-Cor gas exchange system. 02 uptake rates of leaf slices (1-2 mm thick) in the dark were measured at the end of the night period using an 02 electrode (Rank Brothers, Cambridge, England) as previously described (7, 8). The use of respiratory inhibitors (potassium cyanide and salicylhydroxamic acid) and uncouplers for estimating the activity of mitochondrial electron transport pathways in vivo was described earlier (7, 8).

RESULTS AND DISCUSSION Analysis of Leaf Components The self-pollinated tobacco lines selected as haploids for low CO2 survival (19) appeared visually much larger (especially SP422, SP432, and SP45 1) than the control parental genotype Wisconsin-38 nine weeks after growth at 600 to 700 ppm CO2 (Fig. 1). These selected lines also flowered a few days earlier. The only exception was the line SP435, which was more like the control in size and flowering time. The growth parameters were not quantified, but it was evident that total leaf area per plant was much larger in the lines SP432 and SP451 (Fig. 1) and SP422 (not shown). Similarly, the specific leaf dry weight was also higher in these lines (Table I), suggesting that SP lines had a higher total dry weight of the aerial part. Earlier growth assays made under field conditions also showed higher yields of the aerial part (both dry biomass and leaf area) in SP lines (20). The carbon content expressed on a leaf area basis was higher in the selected lines (except SP435; Table I), and the variation of this parameter mainly explained the higher specific leaf dry weight (Table I). The nitrogen and sulfur levels per unit area did not significantly differ among lines (Table I). When expressed on a dry weight basis, the carbon, nitrogen, and sulfur contents either did not significantly change or slightly decreased in some selected lines (Table I). The Chl a/b ratio and the leaf Chl, carotenoid, and alkaloid contents expressed on either an area or dry weight basis were similar in all lines studied, although they were slightly lower in the SP lines (Table II). In this sense, SP451 had significantly lower values of Chl and carotenoids (Table II). These results suggested that large quantitative changes in the amount of some secondary metabolites (e.g. photosynthetic pigments, alkaloids) did not occur in the SP lines, but perhaps these small changes might have contributed to their better growth performance in the long term by saving some energy invested in the production and maintenance of these molecules (see "Leaf Respiration in the Dark" for further discussion). Leaf Photosynthesis and Stomatal Conductance The leaf net CO2 assimilation rates per unit area measured in situ in the growth room at high CO2 concentrations ranged

PHOTOSYNTHESIS AND RESPIRATION IN SELECTED TOBACCO PLANTS

Figure 1. Comparison between the size of two selected tobacco lines (top, SP432; bottom, SP451) and the control line (Wisconsin38) after growing for 9 weeks in a cabinet at 600 to 700 ppm CO2. The pots were 10 cm high. SP422 is not shown but was similar in size to that of SP432 and SP451. SP435 (not shown) was more similar in size to the control line.

from 8 to 11 ,umol. m-2 * s-', being slightly lower in the selected SP lines (Fig. 2). However, the global photosynthesis rate of the selected plants was presumably much higher than in the control genotype because those plants had a much larger total leaf area (Fig. 1). On the other hand, the selected lines had lower values of leaf net photosynthesis per unit dry weight (data not shown). Similar or lower leaf photosynthesis rates were obtained when these selected plants were grown at ambient CO2 levels either in a cabinet or in the field (12, 13). The shape of the curve relating net CO2 assimilation per unit

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leaf area and intercellular CO2 pressure showed no substantial differences among the different lines (Fig. 2). Accordingly, the CO2 compensation point was about 50 ppm under the conditions used in all lines studied. Thus, the evidence suggests that the selection procedure based on survival of haploid plants at CO2 levels slightly higher than the CO2 compensation point (19) did not yield diploid plants with higher photosynthesis rates and significantly lower CO2 compensation points (ref. 12 and E. Delgado, D.W. Lawlor, M. Parry, A.J. Keys, and H. Medrano, unpublished results). Consistently, the specificity factor of Rubisco was unchanged in selected SP lines (E. Delgado, D.W. Lawlor, M. Parry, A.J. Keys, and H. Medrano, unpublished results). Other attempts to select C3 plants with a much lower CO2 compensation point have been unsuccessful (10, 21, 24, 26). Small differences in the CO2 compensation point associated with the component of respiration in the dark (6) may have occurred (see later) but could not be detected with our gas exchange system. The leaf stomatal conductance measured in plants kept in the growth cabinet was lower in the lines SP422, SP432, and SP451 compared with both the control Wisconsin-38 and SP435 (not shown). Changes in conductance were not apparently related to changes in the relative water content and stomatal number: the relative water content was about 78 to 80% in all lines (not statistically different) and the stomatal density and index of the adaxial and abaxial leaf surfaces were very similar or slightly higher in SP45 1 compared with the parental cv Wisconsin-38 (results not shown). The possibility of an improved water economy in the selected lines, which could help the plant to survive under low CO2 conditions (which promote stomata opening), should be further investigated. Leaf Respiration in the Dark

The rates of CO2 release and 02 uptake per unit area of adult tobacco leaves in the dark were about 0.5 ± 0.05 Atmol. m-2s and did not significantly differ among the studied lines (results not shown). However, leaf 02 uptake rates in the dark were lower in the SP lines (especially in SP432 and SP45 1) when expressed on a dry weight basis (Fig. 3) and were negatively correlated with the specific leaf dry weight (Fig. 3A) and with the carbon content per unit area (Fig. 3B). A similar negative relationship between respiration (CO2 release) and specific dry weight was found when these same lines were grown in the field (12). A strong negative relationship between 02 uptake of whole young seedlings (including

Table I. Specific Dry Weight and Carbon, Nitrogen, and Sulfur Contents of Adult Tobacco Leaves Values are means ± SE of 11 to 22 replicates in the case of specific dry weight and of three to four replicates for the other parameters. Asterisk, statistical comparison between the selected and the control (Wisconsin-38) lines is significantly different (P < 0.05). Carbon Sulfur Line Specific Dry Nitrogen % of dry wt % of dry wt % of dry wt 9.m-2 9 .m2 9 .m2 9 .m-2 0.16 ± 0.008 0.52 ± 0.02 39.50 ± 0.20 0.86 ± 0.10 2.81 ± 0.32 30.9 ± 0.9 12.07 ± 0.15 Wisconsin 38 2.67 ± 0.22 0.16 ± 0.011 0.46 ± 0.02 0.90 ± 0.10 13.11 ± 0.64 39.06 ± 1.03 32.6 ± 1.4 SP435 0.17 ± 0.004 0.95 ± 0.05 2.65 ± 0.15 0.47 ± 0.02 14.70 ± 0.36* 40.91 ± 0.41* 34.6 ± 1.1* SP422 2.44 ± 0.30 0.16 ± 0.009 0.39 ± 0.01* 40.09 ± 0.46 0.98 ± 0.14 16.11 ± 0.59* 39.7 ± 1.0* SP432 1.83 ± 0.12* 0.16 ± 0.007 0.37 ± 0.02* 17.41 ± 0.51* 0.78 + 0.05 40.76 ± 0.51 42.9 ± 1.0* SP451


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Table II. Photosynthetic Pigments and Alkaloid Content of Adult Tobacco Leaves Values are means ± SE of four to seven replicates. See Table I legend for other details. Chi Line Carotenoids

Wisconsin38 SP435 SP422 SP432 SP451

a+b

a+b

mg.m-2 330±10 361 ±13 346±11 340±28 300 ± 7*

mg-g-' drywt

mg.m-2

mg 9-1 dry wt

mg.r-2

mg-g1' drywt

10.0±0.8 11.7±0.4 11.0±0.6 9.0±0.7 7.6 ± 0.6*

3.78±0.11 3.72±0.08 3.57±0.03 3.55±0.04 3.64 ± 0.06

49±2 52±2 47±2 44±2 42 ± 1*

1.48±0.12 1.66±0.07 1.51 ±0.09 1.16±0.05 1.06 ± 0.07*

51.4±4.4 45.6±2.8 51.8±4.0 57.3±6.7 63.2 ± 4.4

1.64±0.12 1.45±0.05 1.42±0.10 1.42±0.14 1.48 ± 0.11

the root) and the total plant dry weight in the dark was also found (X. Aranda and J. Azc6n-Bieto, unpublished results). On the other hand, a positive relationship was found between 02 uptake per unit dry weight and leaf nitrogen (Fig. 4A) and sulfur (Fig. 4B) contents (expressed as percentage of dry weight) in the dark. These results suggest that selected SP lines were able to maintain rates of respiration per unit area similar to the control genotype, despite the fact that these lines accumulated more carbon (i.e. carbon compounds) per unit leaf area. Consequently, the rates of respiration expressed on a dry weight or carbon basis were lower in the SP lines. The fact that these plants were grown at high CO2 undoubtedly facilitated carbon accumulation and, perhaps, exaggerated possible genotypic differences in this character. The rate of respiration of leaves in the dark may depend on the carbohydrate content, as shown in several species (2, 5, 6, 8, 16), and this dependence may have varied in the selected tobacco lines. The leaf respiration of selected SP lines was apparently less sensitive to carbon content, allowing a greater carbon accumulation per unit area and, perhaps, more efficient carbon storage for subsequent use in growth. The use of the uncoupler of oxidative phosphorylation CCCP suggested that leaf respiration of the studied lines was controlled by adenylates in a similar

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Figure 2. Net CO2 assimilation rates of adult tobacco leaves at different intercellular C02 levels. Quantum flux density (400-700 nm) was 300 Mmol. m-2. s- . Leaf temperature was 240C.

12 14 16 18 Carbon content (g.m ) Figure 3. Relationship between 02 uptake per unit dry weight (DW) and either specific dry weight (A) or carbon content per unit area (B) in adult tobacco leaves in the dark.

PHOTOSYNTHESIS AND RESPIRATION IN SELECTED TOBACCO PLANTS

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because CCCP stimulated the rate of 02 uptake of leaf slices by about 35 to 45% (results not shown). It is generally considered that some reduction of total plant respiration would offer the possibility of a small, but significant, increase in yield (1, 15, 16). In this sense, several authors (1, 11, 15, 16, 28) suggested that selection of plants for lower mature leaf respiration per unit dry weight results in significantly higher yields. Bugbee and Salisbury (9) discussed the limits of crop potential productivity and suggested that carbon use efficiency, which is inversely related to respiration, can still be significantly increased in plants. However, it is still a way,

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matter of discussion among physiologists and biochemists whether a true "wasteful" component of respiration really exists (3, 5), and this question should be clarified. A possible wasteful component of respiration is the existence of the alternative oxidase, which is the terminal end of a nonphosphorylating mitochondrial electron transport pathway (16). The capacity of the alternative pathway in tobacco leaves, estimated by the cyanide-resistant rate (corrected for any residual respiration), was rather small, being significantly higher in the lines SP422 and SP45 1 (Table III). The percentage of cyanide resistance of total respiration generally increased in the selected lines (see values in parentheses in Table III). However, the alternative pathway was not normally active in these leaves in the absence of cyanide (Table III), even when respiration was stimulated by the uncoupler CCCP (not shown), suggesting that the cytochrome pathway was predominantly used in respiration. Thus, the alternative oxidase of adult leaves seems not to play a significant role in the determination of the carbon balance of the selected tobacco lines in the growth conditions used, but this pathway might have been important for survival during the low CO2 selection period (19). Another possible wasteful component of respiration is a portion associated with "maintenance" processes, not in the sense that reflects an uncoupled respiration (normally the cytochrome path is involved in maintenance respiration) but in the sense that it may support excessively high rates of protein turnover in relation to the growth rate (1, 15). The lines of Lolium perenne selected for low respiration of mature leaves seem to have a reduced maintenance component of respiration (1 1, 15, 28). The respiration rate of the tobacco lines selected by survival to low CO2 seem to differ also in the maintenance component, because the respiration rate was correlated with the nitrogen and sulfur content (Fig. 4), which reflects the protein content. The fact that the maximum differential growth responses in the field between selected and control tobacco lines mainly occur during later growth stages (H. Medrano, unpublished data) is consistent with reduced maintenance respiration, because the biomass that needs to be maintained is larger in older plants. Table l1l. Capacity and Degree of Engagement (p) of the Alternative Pathway in Adult Tobacco Leaves Values are means ± SE of four to 12 replicates (Vf) and of two to three replicates (p). The parameter p is equal to Vaft/Va.,, where Vft is the activity of the alternative pathway. The values in parentheses are the percentage of cyanide-resistant and salicylhydroxamic acid-sensitive rate of total 02 uptake. 02 uptake was measured in thin slices (1-2 mm thick) submerged in a solution containing 10 mM Mes buffer (pH 6.6) and 0.2 mm CaC12. Concentrations of KCN and salicylhydroxamic acid used were 1 and 10 mm, respectively. See "Materials and Methods" and Table I legend for other details. Line

pMOl 02 *m21 * Wisconsin 38 0.06 ± 0.01 (12) 0.06 ± 0.005 (14) SP435 0.14 ± 0.015' (28) SP422 SP432 0.08 ± 0.03 (19) SP451 0.18 ± 0.02* (38)

Va,t AMol 02 *g dry wt * h 7.8 ± 1.5 9.4 ± 0.9 15.5 ± 2.4* 9.7 ± 2.8 16.4 ± 1.4*

p ratio

0 0 0 0 0

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DELGADO ET AL.

Interestingly, the selected tobacco lines have similar respiratory characteristics to those of the selected Lolium lines of

Wilson (28), e.g. a lower respiration per unit dry weight (but not per unit area) of mature leaves, predominance of the cytochrome pathway and no engagement of the alternative oxidase in leaves (even in the presence of uncouplers [11]), and similar adenylate control of respiration (1 1). In conclusion, the apparently improved carbon economy of the tobacco lines selected by survival under CO2 compensation point conditions could be related, at least partially, to a reduced leaf respiration rate (mainly cytochrome pathway) per unit carbon. The reduced respiration rate per unit carbon (see above) could have significantly contributed to the saving of carbohydrates and other substrates during the initial selection period with very low CO2 availability for growth (19), helping to keep a more positive carbon balance and, hence, increase the survival chance under such drastic starvation conditions. The optimization of the carbon balance in the selected tobacco lines compared with the parental genotype Wisconsin-38 could have presumably accentuated the growth differences when grown in conditions favoring large carbon accumulation in the plant, such as the high atmospheric CO2 levels used in this study. ACKNOWLEDGMENTS We thank the Programa Nacional de Investigaci6n Agricola, PLANICYT, Spanish Government, for financial support. We also thank Prof. A. Caballero and Dr. B.G. Drake for critical reading of the manuscript; Maria Reixach and Isidre Casals of the Serveis Cientifico-Tecnics of the University of Barcelona for determinations of carbon, nitrogen, and sulfur; Roser Matamala for stomata number determinations; Xavier Labrafia and Jordi Bort for pigment analysis; and Miquel A. Gonzalez-Meler and Miquel Ribas-Carbo for assistance with the figures.

LITERATURE CITED 1. Amthor JS (1989) Respiration and Crop Productivity. Springer-

Verlag, New York 2. Amthor JS (1991) Respiration in a future, higher-CO2 world: opinion. Plant Cell Environ 14: 13-20 3. ap Rees T (1988) Hexose phosphate metabolism in non-photosynthetic tissues of higher plants. In J Preiss, ed, The Biochemistry of Plants, Vol 2. Academic Press, New York, pp 1-33 4. Ashley DA, Boerma HR (1989) Canopy photosynthesis and its association with seed yield in advanced generations of a soybean cross. Crop Sci 29: 1042-1045 5. Azc6n-Bieto J (1991) Relationships between photosynthesis and respiration in the dark in plants. In J Barber, MG Guerrero, H Medrano, eds, Trends in Photosynthesis Research. Intercept Ltd, Andover, UK 6. Azc6n-Bieto J, Osmond CB (1983) Relationship between photosynthesis and respiration. The effect of carbohydrate status on the rate of CO2 production by respiration in darkened and illuminated wheat leaves. Plant Physiol 71: 574-581 7. Azc6n-Bieto J, Lambers H, Day DA (1983) Respiratory properties of developing bean and pea leaves. Aust J Plant Physiol 10: 237-245 8. Azc6n-Bieto J, Lambers H, Day DA (1983) Effect of photosynthesis and carbohydrate status on respiratory rates and the involvement of the alternative pathway in leaf respiration. Plant Physiol 72: 598-603


9. Bugbee BG, Salisbury FB (1988) Exploring the limits of crop productivity. I. Photosynthetic efficiency of wheat in high irradiance environments. Plant Physiol 88: 869-878 10. Cannell RQ, Brun WA, Moss DN (1969) A search for high photosynthetic rate among soybean genotypes. Crop Sci 9: 840-841 11. Day DA, De Vos OC, Wilson D, Lambers H (1985) Regulation of respiration in the leaves and roots of two Lolium perenne populations with contrasting mature tissue respiration rates and crop yields. Plant Physiol 78: 678-683 12. Delgado E (1990) Caracterizaci6n fotosintetica de lineas de Nicotiana tabacum L seleccionadas en camara de bajo contenido en CO2. Doctoral Thesis. Universitat de les Illes Balears, Palma de Mallorca, Spain 13. Delgado E, Medrano H (1989) Crecimiento y producci6n de genotipos procedentes de haploides de Nicotiana tabacum L seleccionados en camara de bajo contenido en CO2. An Edafol Agrobiol 48: 839-849 14. Elmore CD (1980) The paradox of no correlation between leaf photosynthetic rates and crop yields. In JD Hesketh, JW Jones, eds, Predicting Photosynthesis for Ecosystem Models, Vol 2. CRC Press, Boca Raton, FL, pp 155-167 15. Hay RKM, Walker AJ (1989) An Introduction to the Physiology of Crop Yield. Longman Scientific and Technical, Hong Kong 16. Lambers H (1985) Respiration in intact plants and tissues: its regulation and dependence on environmental factors, metabolism and invaded organisms. In R Douce, DA Day, eds, Encyclopedia of Plant Physiology, New Series, Vol 18. Springer-Verlag, Berlin, pp 418-473 17. Lichtenthaler HK, Wellburn AR (1983) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem Soc Trans 603: 591-592 18. McDermitt DK, Norman JM, Davis JT, Ball TM, Arkebauer TJ, Welles JM, Roemer SR (1989) CO2 response curves can be measured with a field-portable closed-loop photosynthesis system. Ann Sci For 46 (suppl): 416s-420s 19. Medrano H, Primo-Millo E (1985) Selection of Nicotiana tabacum haploids of high photosynthetic efficiency. Plant Physiol 79: 505-508 20. Medrano H, Pol A, Delgado E (1989) Plant production, photosynthesis rate and related characters in doubled-haploid lines of Nicotiana tabacum selected by photosynthetic efficiency. In J Barber, R Malkin, eds, Techniques and New Developments in Photosynthesis Research. Plenum Press, New York, pp 481-484 21. Menz KM, Moss DN, Cannell RQ, Brun WA (1969) Screening for photosynthetic efficiency. Crop Sci 9: 692-694 22. Nasyrov YS (1978) Genetic control of photosynthesis and improving of crop productivity. Annu Rev Plant Physiol 29: 215-237 23. Nelson CJ (1988) Genetic associations between photosynthetic characteristics and yield: review of the evidence. Plant Physiol Biochem 26: 543-554 24. Nelson CJ, Asay KH, Patton LD (1975) Photosynthetic responses of tall fescue to selection for longevity below the CO2 compensation point. Crop Sci 15: 629-633 25. Saunders JA, Blume DE (1981) Quantitation of major tobacco alkaloids by high-performance liquid chromatography. J Chromatogr 205: 147-154 26. Widholm JM, Ogren WL (1969) Photorespiratory-induced senescence of plants under conditions of low carbon dioxide. Proc Natl Acad Sci USA 63: 668-675 27. Wilson D (1973) Physiology of light utilization by swards. In GW Butler, RW Bailey, eds, Chemistry and Biochemistry of Herbage, Vol 2. Academic Press, London, pp 57-101 28. Wilson D (1975) Variation in leafrespiration in relation to growth and photosynthesis of Lolium. Ann Appl Biol 80: 323-338 29. Zelitch I (1982) The close relationship between net photosynthesis and crop yield. BioScience 32: 796-802