83, pp. 8157-8161, November 1986. Botany. Response of two pea hybrids to CO2 enrichment: A test of the energy overflow hypothesis for alternative respiration.
Proc. Nati. Acad. Sci. USA
Vol. 83, pp. 8157-8161, November 1986 Botany
Response of two pea hybrids to CO2 enrichment: A test of the energy overflow hypothesis for alternative respiration (cyanide-resistant respiration/alternative pathway/carbon budgets/Pisum satvum L.)
MARY E. MUSGRAVE*, BOYD R. STRAIN, AND JAMES N. SIEDOW Department of Botany, Duke University, Durham, NC 27706
Communicated by Paul J. Kramer, July 14, 1986
Two pea (Pisum saivum L.) hybrids differing ABSTRACT in the presence or absence of the cyanide-resistant (alternative) pathway of respiration were constructed by reciprocally crossing cv. Alaska and cv. Progress No. 9. The F1 hybrids were grown in greenhouses maintained at either 350 or 650 ppm C02, and the growth, flowering, and dry matter accumulation were compared. The objective was to assess the significance of the alternative respiratory pathway to whole-plant carbon budgets and further to test the hypothesis that the alternative pathway is important in oxidizing excess carbohydrates such as might accumulate under conditions of CO2 enrichment. More carbohydrates were available in the F1 hybrid lacking the pathway, as evidenced by greater plant height, leaf area, specific leaf weight, and total dry matter compared with the reciprocal hybrid, especially at 650 ppm CO2. Specific leaf weight increased markedly under CO2 enrichment in the hybrid lacking the pathway, while it was the same at 350 and 650 ppm in the reciprocal cross. The hybrid lacking the alternative pathway also outperformed the reciprocal cross in terms of total dry matter and seed production. Increased branching with CO2 enrichment was observed in the hybrid lacking the pathway, while branching in the reciprocal cross was only slightly stimulated. These results suggest that alternative respiration consumes luxury carbohydrate and that respiration via this pathway may be considered energetically wasteful in terms of whole-plant carbon budgets.
wheat was germinated in 25% C02/75% air, maximum rates of the alternative pathway in isolated mitochondria increased 2 to 6 times the control value, depending on variety. Lambers (10) and co-workers have suggested that in situations in which excess carbohydrate is produced (such as CO2 enrichment), the nonphosphorylating alternative pathway may contribute significantly to total respiration. AzconBieto et al. (11) have shown a dependence of both respiratory rates and engagement of the alternative pathway on carbohydrate status in spinach and wheat leaves. Oxygen uptake by leaves in the dark was faster after several hours of photosynthesis and involved flux through the alternative pathway. Since supplied sugars could cause the same effect, they concluded that the alternative pathway contributes to total respiration whenever carbohydrate status is high. In a separate study with wheat flag leaves (12), they found leaf respiration rates high (with a cyanide-resistant component) at the beginning of the night; at the end of the night when carbohydrate reserves were low, respiration rates were low and mediated exclusively through the cytochrome (cyanidesensitive) pathway. In a recent report (13), we described two pea cultivars differing in the presence or absence of the alternative pathway. Through hybridization and respiratory analyses of crosses between these two cultivars, we later showed the trait to be maternally inherited (14). F1 hybrids produced by reciprocally crossing the two pea cultivars allow the comparison of growth in plants differing in the presence or absence of the alternative pathway. In the present study, this material was used to investigate the impact of the alternative pathway on the overall carbohydrate budget of the plant.
Three possible responses by plants to the extra carbon fixed under conditions pf CO2 enrichment are accumulation of starch in the chloroplasts (1-3), structural accumulation as more luxuriant growth (4), and higher dark respiration rates. Hrubec et al. (5) grew soybeans under conditions of CO2 enrichment and observed nearly a doubling of dark respiration rates in young leaf tissue (49.3 Amol of CO2 dm 2 hr at 1000 ppm; 25.2 AmoI of CO2-dm 2hr-' at 350 ppm), and Hellmuth (6) reported that dark respiration rates below 320C increased with higher CO2 concentration (140, 300, and 520 ppm). Gifford et al. (7), on the other hand, found root respiration of wheat, sunflower, and mung bean to respond differently to growth under CO2 enrichment depending on the species. In wheat, growth at 680 ppm CO2 was associated with a reduction in root respiration of up to 45%; in mung bean, respiration remained the same; in sunflower, root respiration increased in the enriched atmosphere. A number of workers have shown that not only the quantity but also the quality of respiration changes under CO2 enrichment. Day et al. (8) found that high (10%) CO2 increased the ethylene-induced rise in alternative (cyanide-resistant) respiratory capacity by a factor of 2 in potato tuber mitochondria. Development of the alternative path in etiolated wheat coleoptiles is also affected by high CO2 treatment (9). When
Seed Sources. Seed of Pisum sativum L. cv. Alaska and cv. Progress No. 9 was obtained from Atlee Burpee Seed (Warminster, PA). Hybrid seed was produced by reciprocally crossing the two cultivars following the methods of Gritton (15) as described by Musgrave et al. (14). When cv. Alaska is the maternal parent, the F1 generation has the alternative pathway; when Progress No. 9 is the maternal parent, no alternative pathway is observed in the progeny. Growth Conditions. Plants were grown in gravel/vermiculite (1:1), one to a pot (15 cm), in the Duke University Phytotron greenhouses. Temperatures tracked the 30-year mean Durham high and low temperatures in 15-day intervals, and for experiment 1 (April-June) ranged from 18'C/100C to 260C/19'C, while in experiment 2 (August-October) they ranged from 270C/210C to 17'C/100C (day temperature/night temperature). Plants were watered twice a day: in the morning with modified half-strength Hoagland's solution (16), and in the afternoon with deionized water. CO2 con-
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Abbreviations: AW, dry matter accumulation; NAR, net assimilation rate; RGR, relative growth rate. *To whom reprint requests should be addressed. 8157
MATERIALS AND METHODS
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Botany: Musgrave et al.
Proc. Nati. Acad. Sci. USA 83 (1986)
centrations of 350 ppm and 650 ppm were maintained by a computer-controlled CO2 injection system (17). Growth Analysis. In experiment 1, eight plants per treatment were minimally sampled during growth (two leaflets per plant),- to obtain data on leaf respiration and specific leaf weight, so that total dry matter of the mature plants could be obtained. In experiment 2, 32 seeds were planted for each treatment, and at each sampling time (9, 16, 23, and 57 days after emergence), seven plants were harvested. Leaf area (leaflets and tendrils) was determined by using a LiCor leaf area meter. At each harvest, dry weights of roots, stems, leaves, and any reproductive parts were determined, and the change in dry weight (A&W), relative growth rate (RGR), and net assimilation rate (NAR) were calculated for two harvest intervals as indicated in Fig. 4. Respiration Measurements. Respiration by discs of young leaves sampled immediately after the dark period was measured polarographically with a Clark-type oxygen electrode. Four discs (1-cm diameter) were used for each sample, and cyanide-resistant respiration was determined in 2.0 ml of 0.1 M potassium phosphate buffer (pH 6.9) by adding 2 mM salicylhydroxamic acid in the presence of 2 mM KCN as described (14). The degree of engagement of the alternative pathway (p) is measured by comparing the inhibition obtained by 2 mM salicylhydroxamic acid in the presence and absence of 2 mM KCN. When no inhibition is observed with added salicylhydroxamic acid alone, the pathway is not engaged and p = 0, while when inhibition by salicylhydroxamic acid is the same in the presence or absence of KCN, the pathway is fully engaged and p = 1.
RESULTS Experiment 1. The results obtained in experiment- 1 describe the growth of the two pea hybrids under a normal temperature regime for field-grown peas. At these low temperatures, emergence occurred 7-11 days after planting. The cross lacking the alternative pathway (maternal parent Progress No. 9) emerged some 4 days before the reciprocal cross, which never caught up at 350 ppm CO2 in terms of plant height (Fig. 1 Upper). The same pattern was observed at 650 ppm CO2 (Lower); however, during the course of the growing season the cross with the alternative pathway (maternal parent Alaska) eventually attained the same height as the cross without the pathway. Respiration by leaf discs (Table 1) of the parental plants at 350 ppm followed the pattern reported earlier (14); Progress
I 150
650 ppm C02 ~~~Progress
100,
No. 9
100
Alaska 50
10
20 30 Time after emergence, days
40
FIG. 1. Growth (expressed as height of main shoot) of pea hybrids at 350 and 650 ppm CO2 in experiment 1 (spring temperature regime). Maternal parent is indicated. Error bars are ±SEM (n = 8).
No. 9 totally lacked the alternative pathway, while Alaska had a pathway capacity equivalent to -25% of the uninhibited respiration rate. In Alaska at -650 ppm, this entire capacity contributed to total respiration (p = 1). The hybrids generally resembled their maternal parent with regard to respiration, and engagement of the alternative pathway occurred only in the 650 ppm plants in early leaves (Table 1).
Table 1. Respiration and specific leaf weight of leaf discs from parental pea cultivars and hybrids produced by reciprocally crossing the parental types
Specific leaf
VT Parental cultivar Early leaf Alaska Progress No. 9 Hybrid Early leaf
Vlt
weight
P
350 ppm
650 ppm
350 ppm
650 ppm
350 ppm
ppm
182 191
225 267
25% (2) 0
23% (3) 0
0
1
650
350 ppm
650 ppm
2.3 2.6
2.4
3.5
Alaska 221 189 27% (8) 23% (6) 0 1 2.2 2.6 Progress No. 9 202 173 0 0 2.6 3.7 Leaf subtending pod Alaska 242 211 21% (5) 19%o (2) 0 0 Progress No. 9 244 204 0 0 Respiratory rates were obtained by using leaf discs taken from the third and eighth leaves from the base and are expressed in nmol of 02-g (fresh wt)-l min-1. The maternal parent of the hybrid is indicated. Engagement of the alternative pathway was either complete (p = 1) or absent (p = 0). The capacity of the alternative pathway (Val) is expressed as a percentage of the uninhibited rate (VT). Specific leaf weight (mg-cm-2) was determined from leaf discs from comparable material. Numbers in parentheses are SEM (n - 3).
Botany: Musgrave et al.
Proc. Natl. Acad. Sci. USA 83 (1986)
Interestingly, leaf tissue of the same age with a ready carbohydrate sink (a pod developing in the axil) showed no engagement of the pathway. Measurements of the specific leaf weight (Table 1) support the idea that excess carbohydrate is metabolized via the alternative pathway. The parental cultivar lacking the path-
8
way (Progress No. 9) showed a marked increase in specific leaf weight at 650 ppm CO2 over that obtained at 350 ppm, while cv. Alaska, which has the pathway, did not increase in -specific leaf weight. The hybrids followed their maternal parent with regard to increases in specific leaf weight at 650 ppm; only the cross lacking the pathway displayed a signif8 icant difference. Growth form of the two hybrids was strikingly different in experiment 1, especially as it responded to C02 enrichment. < ,4 As shown diagrammatically in Fig. 2 (Inset), the cross having U the pathway (maternal parent Alaska) branched at the node subtending the first reproductive node, while the reciprocal cross branched exclusively from the base. CO2 enrichment stimulated the development of branches in both hybrids based on the lateral shoot index (L,,; the sum of the length of the laterals; see ref. 18) (Fig. 2). However, the cross lacking the alternative pathway responded more strongly to CO2 4 enrichment than the reciprocal cross with nearly a 4-fold increase in lateral shoot index. Total number of pods formed difered significantly be-tween the hybrids, especially under CO2 enrichment (Fig. 3). It is also interesting to note that flowering and podset began days earlier the 8 cross lacking the alternative pathway 4 days earlier inin the than in the reciprocal cross. Total dry matter accumulation as well as the total seed weight (Table 2) were significantly 4 higher in the cross lacking the pathway. In general, the cross
Progress
.
Alaska 650 ppm
Pr
ogress ppm ~~~~~~~650
PODS 20
350 ppm
800
1-IEl
[0
:
:: I
0
pp650
600
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30 Time after emergence, days
40
FIG. 3. Flowering and podset for pea hybrids grown in experi-
ment 1. The unshaded section of the curve represents the mean number of buds and flowers per plant (n = 8), while the shaded area represents the number of pods (>1 cm). Maternal parent of the cross is specified. Error bars are ±SEM (n = 7).
lacking the pathway showed a greater response to CO2 enrichment than the reciprocal cross. Experiment 2. To further understand the differences in carbon budgets between the two hybrids under conditions of
E
Table 2. Dry matter accumulation by hybrids growing under two
400
L
Alaska
Progress
concentrations of CO2 Seeds Alaska
Progress No. 9
200:
Pods Alaska
Progress No. 9 650
Shoots Alaska
350 350 .: .......Progress No. 9 Roots Alaska Alaska Progress Progress No. 9 Maternal parent Total
FIG. 2. Li. for plants grown under two levels of CO2 (350 or 650 ppm) is expressed as the sum of the lengths of the laterals per eight plants. Maternal parent of the cross is indicated. (Inset) Representative mature architectures for these plants.
Alaska
Progress No. 9
350 ppm
650 ppm
9.2 (0.3) 10.5 (0.5)
9.7 (0.5) 11.4 (0.2)
1.9 (0.1) 2.4 (0.1)
2.1 (0.1) 2.6 (0.1)
4.6 (0.2) 6.1 (0.2)
5.7 (0.3) 6.5 (0.1)
1.1 (0.1) 1.4 (0.1)
1.3 (0.1) 2.1 (0.1)
17.4 (0.8)
18.8 (0.8)
20.4 (0.8) 22.6 (0.4) Maternal parent is indicated. Results are expressed as g (±SEM); n = 8.
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0
Botany: Musgrave et A
Proc. Natl. Acad. Sci. USA 83 (1986)
100
)0 |
to
Alaska (D
O
50
20
40 20 40 Time after emergence, days
60
FIG. 4. Growth of pea hybrids at 350 and 650 ppm CO2 in experiment 2. Arrows represent harvest times in the growth analysis; error bars are ±SEM (n = 7). Maternal parent is indicated. Harvest intervals 1 and 2 are shown.
CO2 enrichment, a growth analysis was undertaken. By planting seeds in warm conditions, emergence was accelerated and occurred 4 days after planting in both hybrids under both CO2 regimes. Three weekly harvests were made (Fig. 4), and a final harvest was made when all treatments had stopped growth (57 days after planting). At this time, no foliage had been lost and the pods were mature in all the treatments except for the hybrid having Progress No. 9 as a maternal parent (no alternative pathway) grown at 650 ppm. These latter plants had continued to produce flowers and therefore had a number of immature pods at the final harvest. AW, NAR, and RGR are tabulated for the two harvest intervals in Table 3. Using the ratio of values obtained at 650 and 350 ppm CO2 as an indication of the increase in the growth parameters attained under CO2 enrichment, hybrids with Progress No. 9 as a maternal parent had large increases in AW, NAR, and RGR, while the reciprocal cross did not. Data on total dry weight, leaf area, specific leaf weight, and seed dry weight at the final harvest are shown in Table 4. Again, the Progress No. 9 maternal parent hybrid outperformed the reciprocal cross in dry weight and showed a large increase in dry weight under 650 ppm CO2. A similar trend was also seen with total leaf area. Specific leaf weight, although not significantly different between the hybrids at 350 ppm C02, did show an increase under CO2 enrichment, as was seen in experiment 1, with Progress No. 9 but not Alaska as the maternal parent. In contrast to experiment 1, in which node of branching varied with the maternal parent of the cross, in experiment 2, all branching occurred at the node subtending the first fertile
Table 4. Final harvest data for pea hybrids grown under two levels of CO2 in experiment 2 350 ppm 650 ppm P Total dry weight, g Alaska 11.1 (0.5) 13.1 (0.9) 99O 12.6 (0.6) Progress No. 9 17.4 (0.6) 99% P 9O 99%O Leaf area, cm2 Alaska NS 449 (22) 479 (38) Progress No. 9 516 (40) 717 (30) 99%o P 95% 99%0 Specific leaf weight, mgcm-2 NS Alaska 2.73 (0.1) 2.70 (0.1) 3.46 (0.2) Progress No. 9 2.94 (0.2) 95% P NS 99O Seed dry wt, g 99% Alaska 5.2 (0.2) 6.2 (0.4) 99% Progress No. 9 5.4 (0.4) 7.0 (0.4) P NS 99%0 Results are expressed as mean (±SEM); maternal parent is indicated. Significance of difference for values between CO2 levels and between hybrids is expressed as a probability value (P) obtained from a t test for paired comparisons. NS, not significant.
node. As before, the hybrid with Progress No. 9 as the maternal parent showed a more substantial response to CO2 enrichment in terms of lateral bud development (Lax increased from 5 cm at 350 ppm to 88 cm at 650 ppm) than the reciprocal cross (Lo increased from 0 to 29 cm).
DISCUSSION Bahr and Bonner (19) first postulated that the alternative pathway may be active in mitochondria only when the capacity of the main cytochrome pathway is exceeded. Palmer (20) and later Lambers (10, 21) extended this concept to the whole plant level and Azcon-Bieto et al. (11, 12) have found that this energy overflow function of the pathway can be evoked in the diurnal respiratory cycles of a number of plants. When plant leaf carbohydrate status was high (at the beginning of the dark period after several hours of photosynthesis) the activity through the alternative pathway was high, while at the end of the night, activity through the pathway was low. Since CO2 enrichment commonly results in increased carbon assimilation, it represents a convenient way of imposing conditions for energy overflow on plants. The respiration results obtained with hybrids having Alaska as a maternal parent (Table 1) support the idea that the alternative pathway operates when carbon reserves are high, since only at 650 ppm is the pathway engaged at the end of the night. Interestingly, in a leaf subtending a developing pod, no engagement of the pathway was observed even at 650 ppm,
Table 3. Summary of growth analysis data for two harvest intervals (I) in experiment 2 Progress No. 9 Alaska Enrichment ratio Growth 350 650 350 650 I ppm ppm ppm ppm Alaska parameter Progress AW, g 1 0.41 0.69 0.35 0.40 1.71 1.15 2 0.87 1.63 1.06 1.29 1.88 1.22 NAR, g-dm2-day-' 1 0.070 0.11 0.066 0.079 1.57 1.20 2 0.062 0.093 0.077 0.090 1.50 1.17 1 RGR, g-g-1day0.123 0.168 0.130 0.130 1.37 1.00 2 0.114 0.138 0.147 0.154 1.21 1.05 and ratios of values obtained for these AW, NAR, RGR, parameters at 650 ppm versus 350 ppm CO2. Maternal parent of the hybrid is indicated.
Botany: Musgrave et al. suggesting that sink relationships can influence the disposition of excess carbohydrate. Lambers (10) reported alternative respiration in storage roots to be higher in the young taproots than later when development of storage capabilities made the root available as a sink. Data on specific leaf weight support the above observation. Whether leaf discs of mature leaves from young plants (Table 1, experiment 1) or entire leaves from mature plants (Table 4, experiment 2) are compared, the hybrid without the alternative pathway showed increases with CO2 enrichment, while the reciprocal cross did not. Paez (22) found no significant difference in specific leaf weight between Alaska peas grown at 350 and 675 ppm CO2 in growth chambers, as would be expected since this parental cultivar carries the alternative pathway. Paez also found CO2 enrichment resulted in no difference in dry matter accumulated by the whole plants. In the present study, no significant difference in dry matter accumulation was found for the Alaska maternal parent hybrid in experiment 1 (Table 2), although a small but significant difference was found in experiment 2 (Table 4). In both experiments 1 and 2, the dry matter accrued by the plants was increased by CO2 enrichment when the alternative pathway was lacking. Thus, both storage carbohydrate (as inferred by specific leaf weight) and structural carbohydrate (leaf area, height, total dry weight) increased under CO2 enrichment in the hybrid lacking the alternative respiratory pathway. Previously, Hardy and Havelka (23) observed CO2 enrichment (1000-1500 ppm) effects in different legumes: including more dry matter production, more rapid vegetative growth, and more seeds. These increases were attributed to decreased photorespiration, delayed senescence, increased plant density, and retention of more reproductive structures. Mauney et al. (24) obtained positive responses to C02-enriched (630 ppm) air with respect to total dry matter production: 382% for soybean, 109%o for cotton, 60% for sunflower, and 18% for sorghum. Kramer (25) has suggested that the relatively small increases in dry matter obtained for sunflower and sorghum reflect the determinate nature of growth in these plants. The present study demonstrates the striking difference in response to CO2 enrichment that can be obtained merely by eliminating the cyanideresistant respiratory pathway. The different- growth forms obtained in experiment 1 (Fig. 2) could be interpreted as a further manifestation of an altered carbon budget due to elimination of the alternative pathway. General nutrition has long been known to regulate lateral bud proliferation (26). The striking effect of CO2 enrichment on branching in the hybrid lacking the pathway in experiment 1 and the much diminished effect on branching in experiment 2, when the plants were growing rapidly, suggests that nutritional status is probably involved. Although all plants in experiment 2 were the same height at the times of the three harvests in the growth analysis, the difference in their carbon economies, which became obvious in terms of height at maturity, was detected in A W, NAR, and RGR. Only the hybrid lacking the alternative pathway showed large increases in these three measures with CO2 enrichment (Table 3). In the first experiment, when the plants were growing more slowly because oflower temperature, this additional assimilate may have permitted the proliferation of basal laterals. Kuiper (27) suggested that the amount of alternative pathway in Plantago major roots was inversely proportional to several indices of vigor. He found a line with slow root growth and few seeds per capsule to have a higher proportion of alternative respiration than a line with faster root growth and many seeds per capsule. The present report draws the same conclusion using plants having a uniform genetic background but differing in the presence or absence of the
Proc. Natl. Acad. Sci. USA 83 (1986)
8161
alternative pathway. In addition, these results suggest that carbon losses to the alternative pathway may become more important as global CO2 levels increase. While total dry matter increased only 14% at 350 ppm when the alternative pathway was eliminated, it increased 33% at 650 ppm (Table 4, experiment 2). In conclusion, this comparison of pea hybrids differing in the maternally inherited character of expressing cyanideresistant respiration shows that plants lacking the alternative pathway outperform those having the pathway in terms of total dry matter production and overall growth, especially under conditions of CO2 enrichment. Storage of nonstructural carbohydrate in the cross lacking the pathway was inferred from the increased specific leaf weight under 650 ppm C02; the reciprocal cross showed no such increase. These results support the hypothesis that cyanide-resistant respiration can consume luxury carbohydrates and that respiration via this pathway may be considered energetically wasteful in terms of whole plant carbon budgets. We thank Drs. David Patterson and Andrew Hanson for helpful discussions, Mary Alice VanHoy for technical assistance, and Prof. W. L. Culberson for providing departmental financial support. This work was supported by National Institutes of Health Grant GM26095 to J.N.S. and National Science Foundation Grants BSR82-15533 and BSR83-14925 to the Duke University Phytotron. M.E.M. acknowledges support from a James B. Duke Graduate Fellowship and the National Institutes of Health Cell and Molecular Biology training grant (T32-GM07184). 1. Cave, G., Tolley, L. C. & Strain, B. R. (1981) Physiol. Plant. 51, 171-174. 2. Wulff, R. & Strain, B. R. (1982) Can. J. Bot. 60, 1084-1091. 3. Sasek, T. W., DeLucia, E. & Strain, B. R. (1984) Plant Physiol. 75, S-6. 4. Sionit, N., Strain, B. R. & Hellmers, H. (1981) J. Agric. Sci. 79, 335-339. 5. Hrubec, T. C., Robinson, J. M. & Donaldson, R. P. (1984) Plant Physiol. 75, S-158. 6. Hellmuth, E. 0. (1971) Photosynthetica 5, 190-194. 7. Gifford, R. M., Lambers, H. & Morison, J. I. L. (1985) Physiol. Plant. 63, 351-356. 8. Day, D. A., Aaron, G. P., Christofferson, R. E. & Laties, G. G. (1978) Plant Physiol. 62, 820-825. 9. McCaig, T. N. & Hill, R. D. (1977) Can. J. Bot. 55, 549-555. 10. Lambers, H. (1980) Plant Cell Environ. 3, 293-302. 11. Azcon-Bieto, J., Lambers, H. & Day, D. A. (1983) Plant Physiol. 72, 598-603. 12. Azcon-Bieto, J., Day, D. A. & Lambers, H. (1983) Plant Sci. Lett. 32, 313-320. 13. Musgrave, M. E. & Siedow, J. N. (1985) Physiol. Plant. 64, 161-166. 14. Musgrave, M. E., Murfet, I. C. & Siedow, J. N. (1986) Plant Cell Environ. 9, 153-156. 15. Gritton, E. (1980) in Hybridization of Crop Plants, eds. Fehr, W. R. & Hadley, H. H. (American Society of Agronomy, Madison, WI), pp. 347-356. 16. Downs, R. J. & Hellmers, H. (1975) Environment and the Experimental Control of Plant Growth (Academic, London). 17. Hellmers, H. & Giles, L. J. (1979) in Controlled Environment Guidelines for Plant Research, eds. Tibbitts, T. W. & Kozlowski, T. T. (Academic, New York), pp. 229-234. 18. Andersen, A. S. (1976) Physiol. Plant. 37, 303-308. 19. Bahr, J. T. & Bonner, W. D., Jr. (1973) J. Biol. Chem. 248, 3441-3445. 20. Palmer, J. M. (1976) Annu. Rev. Plant Physiol. 27, 113-157. 21. Lambers, H. (1985) in Encyclopedia of Plant Physiology (Springer, Berlin), Vol. 18, pp. 418-473. 22. Paez, A. B. (1982) Dissertation (Duke University, Durham, NC). 23. Hardy, R. W. F. & Havelka, U. D. (1977) in Biological Solar Energy Conversion, eds. Mitsui, A., Miyachi, S., San Pietro, A. & Tamura, S. (Academic, New York), pp. 299-322. 24. Mauney, J. R., Fry, K. E. & Guinn, G. (1978) Crop Sci. 18, 259-263. 25. Kramer, P. J. (1981) BioScience 31, 29-33. 26. Gregory, F. G. & Veale, J. A. (1957) Soc. Exp. Bot. 11, 1-20. 27. Kuiper, D. (1983) Physiol. Plant. 57, 222-230.
