Whole Plant Studies on Photosynthesis and Acetylene Reduction in Alnus glutinosa

Whole Plant Studies on Photosynthesis and Acetylene Reduction in Alnus glutinosaAuthor(s): J. C. Gordon and C. T. WheelerSource: New Phytologist, Vol. 80, No. 1 (Jan., 1978), pp. 179-186Published by: Wiley on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/2431649 .

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New Phytol. (1978) 80,179-186.

WHOLE PLANT STUDIES ON PHOTOSYNTHESIS AND ACETYLENE REDUCTION IN ALNUS GLUTINOSA

By J. C. GORDON* and C. T. WHEELER

Department of Botany, University of Glasgow, Glasgow

(Received 27 May 1977)

SUMMARY

Rates of photosynthesis and acetylene reduction by whole plants were measured on (a) twelve Alnus glutinosa clones and (b) A. glutinosa seedlings grown in low and high irradiances. Both genetic and environmental manipulation of photosynthetic rates produced concomitant changes in rates of acetylene reduction. For t-he clonal material, growing under closely similar environmental conditions, mean rate of net photosynthesis was significantly positively correlated with both the mean fresh weight of nodules formed per plant and with the mean rate of acetylene reduction.

INTRODUCTION

Previous studies of the relationship between carbohydrate supply and symbiotic nitrogen fixation in alder have relied on assays of acetylene reduction by detached nodulated roots for the estimation of nitrogenase activity (Wheeler, 1971 ; Wheeler and Lawrie, 1976). Subse- quent work has shown that not only is the rate of acetylene reduction decreased by the detopping procedures used in earlier experiments but also that the decrease in acetylene reduction does not always bear a constant relationship to the nitrogenase activity of the whole plant (Wheeler, Cameron and Gordon, 1978). These findings suggest that it would be of value to re-examine earlier data, using whole plants in the acetylene reduction assays, and also to include simultaneous measurements of photosynthetic rates by infra-red gas analysis. The omission of direct measurements of photosynthetic rate in previous studies of the dependence of nodule function on carbohydrate supply in non-legume species has prevented quantitative correlation of rates of carbon and nitrogen fixation, although recent studies on inter-relationships between photosynthesis, water potential and acetylene reduction in soybean have employed gas exchange techniques for measurements of photosynthetic rate (Huang, Boyer and Vanderhoef, 1975).

In alder (Wheeler, 1971; Wheeler and Bowes, 1974; Wheeler and Lawrie, 1976) as in other species (see Gibson, 1976; Sprent, 1976; Hardy and Havelka, 1976), rates of nitrogen fixation or acetylene reduction are affected by treatments, such as shading, darkening or defolia- tion, which would be expected to have their greatest impact initially on photosynthesis. However, the rapidity of the response to changes in irradiance varies with species and de- pends also on plant size and development. Thus, although Akkermans (1971) found no significant changes over a 24-h period in the nodules of large, field-grown Alnus glutinosa, Wheeler (1969, 1971) reported significant diurnal fluctuations and a fairly rapid response

* P'resent address: Department of lorest Science, Oregon State University, Corvallis, Oregon 97331, U.S.A.

179



180 J. C. GORDON and C. T. WHEELER

(within 12 h) to continual darkness in young, glasshouse-grown alders. In young alder plants, radio-tracer experiments have shown that a small portion of the photoassimilate supply is translocated rapidly to the nodules, although it is not clear whether it can support nitrogenase activity (Wheeler and Lawrie, 1976).

In the present study, whole plants and infra-red gas analysis have been used to assay acetylene reduction and photosynthesis of young alder plants. To avoid possible effects of surgical treatments on acetylene reduction (Wheeler et al., 1978), both genetic and environmental methods have been employed to modify rates of photosynthesis in experiments de- signed to explore further relationships between carbon and nitrogen fixation in this species.

MATERIAL AND METHODS

Two sets of young plants of A lnus glutinosa (L.) Gaertn. were used. To produce plants with a high probability of exhibiting genetically based differences in photosynthetic capacity, many individual plants were grown from seed in the glasshouse under the same general cultural regime and environment (Bajuk, Gordon and l'romnitz, 1977). The plants were not inoculated, did not nodulate and were well supplied with combined nitrogen. Individuals showing high and low rates of growth in the favourable environment were vegetatively propagated from single-internode, greenwood cuttings and grown on in a growth chamber under 16 h, 20?C days and 8 h, 140C nights. Several ramets from each of twelve selected individuals were so grown to produce twelve clonal lines. Each ramet, after being rooted under mist, was inoculated with A. glutinosa endophyte from a single preparation of crushed- nodule inoculum.

After 8 weeks growth in the controlled environment chamber, rates of photosynthesis and acetylene reduction were measured on all clonal plants as described below and dry weights of plant parts were obtained.

The second group of plants was grown from seed in the glasshouse. After 10 weeks growth, the tops of the plants were removed and the detopped plants placed in one of two controlled environments: one with a higher irradiance (30 W m-2 photosynthetically active radiation-l'AR) and one with lower irradiance (8 W m-2 PAR). The new tops, which arose quickly from suppressed buds just above the root collar, were grown on for a further 10 weeks in the new light environment and then ten plants from each light environment were used for the measurements of rates of photosynthesis and acetylene reduction as described below. The use of cutback plants avoided the influence of lighting conditions on the pro- cesses of infection and early nodule development in seedlings, which are probably considerable. Some nodule necrosis occurred during the regrowth of the tops, particularly in plants subjected to the lower irradiance, and only healthy nodules were included in the final weighing.

Rates of photosynthesis were measured on whole plants in a specially constructed 12-1 plexiglass container placed inside the growth chamber in which the alders were grown. An open-circuit system was used to monitor CO2 concentration in the chamber. It included, in series, these elements: 1. A compressed air source of known (c. 320 ppm) CO2 concentration. 2. A humidification system comprising two constant-temperature water baths, one at 140C and one at 200C. Air from the source was saturated with water vapour in the 140C bath and then passed through a copper coil in the 200C bath, producing an air stream with a constant dewpoint of 140C. 3. The reference cell of a Grubb-Parsons differential infra-red CO2 analyser. 4. The plexiglass chamber into which the alder to be measured was sealed.



Photosynthesis and acetylene reduction in alder 181

Temperature during measurement was always within ? 1?C of growth chamber temperature. 5. The sample cell of the CO2 analyser. 6. A Rotameter flow rate meter. A flow rate of 21 min-' which was sufficient completely to change chamber air every 6 min was maintained.

After preconditioning in the chamber for 45 min, the rate of CO2 exchange was moni- tored for each plant for at least 1 h, during which time rates were stable, indicating a constant plant response to the experimental environment. For a set of plants from each light environment, photosynthetic responses to light intensity were determined using the two experimental irradiances (8 and 30 W m 2 PAR) and one lower irradiance (1.5 W m 2 PAR). Rates of acetylene reduction were measured by enclosing whole plants, undisturbed in the peralite-filled plastic pots in which they were grown, in plastic bags closed around subaseal stoppers with a twistum. Acetylene (0.51) was then injected into the 4-1 bags and 1-ml samples were withdrawn at 1 5-min intervals and analysed for ethylene. Empty bags and bags containing killed root systems in peralite and plastic pots were included as controls. A separate series of tests was conducted to determine rates of acetylene and ethylene leakage from the bags and one set of plants was measured both by the bag method as above and by enclosing only the pots in specially constructed glass jars. Rates determined by the two methods agreed closely.

RESULTS

Clonal coinparisons In addition to differing in growth, the alder clones differed in rate of net photosynthesis

per plant, rate of acetylene reduction per plant and in rate of acetylene reduction per g nodule fresh weight (Table 1). The clones did not differ, however, in rate of photosynthesis per dM2 leaf area (Table 2). The variance between clones in the rate of acetylene reduction per g nodule fresh weight (i.e. nodule efficiency) was not significantly different from the variance in acetylene reduction expressed on a per plant basis (Table 2). It is not possible to infer, therefore, whether nodule efficiency or the capacity of clones to form nodules was

Table 1. Rates of photosynthesis and acetylene reduction, nodule fresh weight, and total plant dry weight means for each of twelve A Inus glutinosa clones

Clone numbers are for identification only, with the first number designating the seedlot and the second the individual plant from which the clone was propagated.

Each tabular value is the mean of three observations.

Acetylene reduction (nmole h1 -) IPhotosynthesis Noduile fresh Total plant

Clone per g fresh per plant weight dry weight (number) per plant wt nodules (mg CO2 h-1) (g) (g)

5-50 335 386 12.8 0.98 5.29 3-25 266 473 10.9 0.55 4.30 3-23 188 533 8.8 0.40 3.79 1-23 179 332 9.3 0.54 4.28 2-50 157 161 11.8 1.02 4.89 2-58 151 275 8.8 0.52 4.10 3-13 142 352 2.9 0.26 2.11 6-15 133 228 6.8 0.58 3.25 6-12 127 232 7.6 0.57 3.21 5-37 123 171 8.6 0.67 3.05 4-40 38 276 6.6 0.14 2.81 3-21 25 92 6.5 0.15 2.79




more important in explaining the observed differences in whole plant acetylene reduction rates.

Net photosynthesis rate per plant correlated well with acetylene reduction rate per plant and with nodule fresh weight per plant for the twelve clones (Table 1). The correlation co- efficients with acetylene reduction (r = 0.59) and with nodule fresh weight (r = 0.68) were both highly significant (P<0.01). Clones 1-13 and 2-50 showed anomalous responses to the observed rates of photosynthesis, however. Clone 3-13 had a nearly average rate of acetylene reduction per plant but a low fresh weight of nodules and the lowest rate of net photosynthesis per plant. Clone 2-50, which also showed a nearly average rate of acetylene

Table 2. Analysis of variance fbr twelve Alnus glutinosa clones upon which rates of acetylene reduction, per plant and per gram nodule fresh weight, and rates of net photosynthesis, per

plant and per gram leaf dry weight, were measured Variable Sources of variation d.f. MS F

Acetylene reduction per plant B3etween clones 11 21,645 3.36** Within clones 24 6,450

Photosynthesis per plant Between clones 11 21.72 5.78** Within clones 24 3.76

Acetylene reduction per unit nodule wt Between clones 11 50,398 2.67* Within clones 24 18,847

Photosynthesis per unit leaf wt Between clones 11 3.78 1.07 Within clones 24 3.54

F** 0.01 (11, 24) 3.09. fi-* 0.025 (1 1, 24) = 2.59.

reduction, had the highest fresh weight of nodules and the second highest rate of net photosynthesis per plant. The total dry weight accumulation of these two clones paralleled their rate of net photosynthesis (Table 1) so that it is unlikely that error in measurement of photosynthesis rate was responsible for the anomalous responses. It would appear, therefore, that nodule efficiency differed greatly between these two clones, with the rate of acetylene reduction per g nodule fresh weight of clone 2-50 less than half that of clone 3-13. Nodule efficiency of clone 3-13 was in the middle of the range of nodule efficiency exhibited by the twelve clones and thus it seems that this clone may allocate a greater percentage of its total photosynthetic product to the nodules, once they are formed, than do most others included in this experiment. Conversely, clone 2-50 may have allocated a much smaller pro- portion of its total photosynthetic product to its nodules after their formation. Studies employing radioactive tracers will be necessary to resolve this question.

These twelve clones, taken together, showed roughly a ten-fold difference in mean acetylene reduction rate per plant and a two-fold difference in mean rate of net photosynthesis per plant. The results suggest that, provided endophyte effectivity is constant, selection of clones for high photosynthetic capacity, as evidenced by dry weight accumulation or CO2 exchange measurements, would usually result in the selection of clones with a high acetylene reduction rate and, presumably, a high nitrogen fixation capacity.

Irradiance comparisons Alders from a mixed population of plants which had been detopped and allowed to re-

grow in irradiances of either 8 or 30 W m-2 differed markedly in growth characteristics




(Table 3). Plants grown under the higher irradiance had approximately 50% greater leaf area and more than twice the total leaf dry weight. Thus, the low irradiance plants had about twice the specific leaf area (dm2 g-1) and half the specific leaf weight (g din2) of the high irradiance alders. Total shoot dry weight of the high irradiance alders was three times those grown under low irradiance but the former plants had only 70% more nodule dry weight,

Table 3. Oven dry weights and leafJarea ofJA lnus glutinosa plants grown at two irradiance levels

Fach tabular value is the mean of ten observations.

Irradiance Oven dry wt (g) Specific during growth Leaf area leaf area (W m 2 PAR) Top Leaves Nodules (dM2) (dM2 g-1)

30 1.58 1.10 0.15 2.38 2.18 8 0.52 0.39 0.10 1.65 4.19

reflecting the influence of the similar light environment under which the root systems of both groups were formed initially.

The photosynthetic response to changes in irradiance of the two groups followed the pattern often reported previously for plants grown at different irradiance levels. Plants grown under 8 W mn2 had a lower rate of dark respiration, a lower apparent light compensation point and a lower rate of net photosynthesis (per plant and per unit leaf area) at the higher (30 W m 2) irradiance. At 8 W rn2, rates of photosynthesis per plant of both high and low light-grown plants were nearly equal and rates per unit leaf area exactly equal (Table 4).

Acetylene reduction rates, expressed on both a 'per plant' and a 'per g nodule dry weight'

Table 4. Rates of net CO2 exchange (- denotes net CO2 release) at three irradiance levels and in the dark fbr alder plants grown at

-high (30 W m 2) and low (8 W m 2) irradiances All CO2 exchange values are the means of four observations.

Irradiance (WX m2 PAR) CO2 exchange (mg CO2 h-1) D)uring growth l)uring assay per plant per dm 2 leaf area

30 0 -41.73 -0.60 1.5 -0.71 --0.46 8 2.04 0.70

30 5.18 1.81 8 0 -0.32 -0.14

1.5 0.12 0.05 8 1.61 0.71

30 2.71 1.20

basis, were increased 45 min after transfer of low light grown plants to higher irradiance levels, although the effect on ethylene production was considerably less than that noted between plants grown continually in the two irradiances (Table 5). A decrease in the rate of acetylene reduction also followed the transfer of plants, grown under the higher irradiance, to the lower irradiance levels, although the change in activity was much less than that which followed transfer of low-irradiance-grown plants to the higher irradiance conditions.



184 J. C. GORDON and C. T. WHiEELER

ITable 5. Rates of/acetylene reduction for Alnus glutinosa plants grown either under low (8 W m 2) or high (30 W m-2) irradiances compared with the rate of acetylene reduction observed 45 min after transfer to

either the higher or lower irradiances, respectively Acetylene reduction in 2 irradiance levels

Irradiance (nmoles C21-2 h) -l) during growth per plant per g nodule dry wt (WV m2 PAR) 8(W -2)30 8(W -2)30

30 136 153 858 1009 8 40 62 437 698

DISCUSSION

The importance of a supply of host-derived photosynthetic products for symbiotic nitrogen fixation has long been recognized (eg. Lindstroim, Newton and Wilson, 1952; Virtanen, Moisio and Burris, 1955; Bach, Magee and Burris, 1958) and their importance in Alnus glutinosa has been demonstrated indirectly (Wheeler, 1969, 1971; Wheeler and Bowes, 1974; Wheeler and Lawrie, 1976). The positive correlations, demonstrated here for twelve different clones, between genetic variation in photosynthetic capacity and both whole plant acetylene reduction and nodule fresh weight per plant (Table 1) provide direct evidence that carbon and nitrogen fixation are closely connected in young alder plants. This conclusion is supported further by the higher whole plant rates of photosynthesis and acetylene reduction found in the higher light conditions when detopped plants were allowed to regrow in either high or low irradiance levels (Table 4). Photosynthesis per unit leaf area and acetylene reduction per g nodule fresh weight were also both higher in high light plants, although the differences were smaller than when expressed on a 'per plant' basis. Reduced acetylene reduction rates in low light plants can be viewed therefore as a consequence of lower photosynthesis (less leaf surface and less photosynthesis per unit leaf surface) and thus lower nitrogenase activity (lower nodule activity, fewer nodules). These findings support the contention of Hardy and Havelka (1976), based on photosynthetic enhancement in soybean through increased CO2 concentration, that supply of photosynthate is a major limiting factor in nitrogen fixation.

A fairly rapid response of nitrogenase activity in young alder plants to changes in supply of photosynthate was suggested by Wheeler (1971) as a result of the decrease in acetylene reduction which followed stein girdling. Although it is now thought possible that handling effects may have contributed to the decrease in nitrogenase activity observed in these experiments (Wheeler, Cameron and Gordon, 1978), further evidence in support of a close connection between whole plant rates of photosynthesis and acetylene reduction was obtained here from the changes in acetylene reduction rate observed 45 min after transfer of plants to different lighting conditions (Table 4). The larger changes in acetylene reduction rate observed when plants were transferred from low to high irradiance, rather than from high to low, supports the contention that the internal carbohydrate status of the plant (i.e. levels of soluble and 'reserve' carbohydrates) may have considerable influence on the response of nitrogen fixation to changed lighting conditions (Wheeler and Lawrie, 1976). Of relevance to this suggestion is the finding that rates of acetylene reduction in soybean are




positively related to the concentrations of sucrose and (+)-pinitol in the nodules (Streeter and Bosler, 1976).

Small, young alder plants, grown in controlled environments in irradiances which were low in comparison to those normally experienced in the field, were used in the experiments reported here. It is probable that as trees become larger and are subjected to more variable environments, the supply of photosynthate to nodules may become much more complex, with more pools involved and greater lags between alterations to photosynthetic rate and consequent effects upon nodule function (Akkermans, 1971; Wheeler and Lawrie, 1976). It is probable, however, that the major principle will hold: that is, that trees genetically dis- posed to produce and distribute to the roots the largest quantities of photosynthate will be most active in nitrogen fixation. This fact, if supported by field experimentation, has importance for practical tree improvement programmes in two ways. Firstly, if it is desired to select alders for highi rates of nitrogen fixation, as well as for growth, an initial selection for high rates of dry matter accumulation should usually produce a population having both attri- butes. Secondly, if enhancement of photosynthesis through a combination of genetic and cultural manipulations is to be practised to increase dry mass yield, then applying those procedures to species capable of symbiotic nitrogen fixation should produce the greatest improvement in yield.

ACKNOWLIE)GMENT

We should like to thank Professor G. Bond for his informed discussion of the work reported here and criticism of the manuscript.

REFI,ERIE,NCI-ES

AKKERMANS, A. 1). L. (1971). Nitrogen fixation and nodulation of Alnus and Hippophae under natural conditions. Ph.). thesis, University of Leiden.

BACH, M. K., MAGEE, W. E. & BURRIS, R. H-. (1958). Translocation of photosynthetic products to soybean nodules and their role in nitrogen fixation. PI. Physiol., Lancaster, 33, 118.

BAJUK, L. A., GORDON, J. C. & PRONITZ, L. C. (1977). FEvaluation of the growth potential of Alnus glutinosa clones. Iowva Stat. Jour. Sci. (in press).

GIBSON, A. H. (1976). Recovery and compensation by nodulated legumes to environmental stresses. In: Symbiotic Nitrogen Fixation in Plants. I.B.P. 7, 385 (E'd. by P. S. Nutman). Cambridge University Press, London.

HlARDY, R. W. 1. & HAVFELKA, U. D. (1976). Photosynthate as a major factor limiting nitrogen fixation by field grown legumes, with emphasis on soybeans. In: Symbiotic Nitrogen Fixation in Plants. I.B.P. 7, 421 (E'd. by P. S. Nutman). Cambridge University Press, London.

HUANG, C-Y., BOYER, J. S. & VANI)ERHOEF, L. N. (1975). Limitation of acetylene reduction (nitrogen fixation) by photosynthesis in soybean having low water potentials. PI. Physiol., Lancaster, 56, 228.

LINDSTROM, F. S., NFEWTON, J. W. & WILSON, P. W. (1952). The relationship between photosynthesis and nitrogen fixation. Proc. nat. Acad. Sci. (U.S.), 38, 392.

SPRFENT, J. I. (1976). Nitrogen fixation by legumes subjected to water and light stresses. In: Symbiotic Nitrogen Fixation in Plants. I.B.P. 7, 405 (Ed. by P. S. Nutman). Cambridge University Press, London.

STE'WART, W. l). P. (1962). A quantitative study of fixation and transfer of nitrogen in Alnus. J. exp. Bot. 13, 250.

STRE'ETE'R, J. G. & BOSLER, M. E. (1976). Carbohydrates in soybean nodules: identification of com- pounds and possible relationships to nitrogen fixation. PI. Sci. Lett. 7, 321.

VIRTANEN, A. I., MOISIO, T. & BURRIS, R. H. (1955). Fixation of nitrogen by nodules excised from illuminated and darkened pea plants. Acta Chem. Scand. 9, 184.

WHF'ELER, C. T. (1969). The diurnal fluctuation in nitrogen fixation in the nodules of Alnusglutinosa and Myrica gale. New Phytol. 68, 675.




WHE-I[:LE'R, C. T. (1971). The causation of the diurnal changes in nitrogen fixation in the nodules of Alnus glutinosa. iVeiv Phytol. 70,487.

WHI::E'LER, C. T. & BOWE'S, B. G. (1974). E`ffects of light and darkness on nitrogen fixation by root nodules of Alnusglutinosa in relation to their cytology. Z. Pflanzenphysiol. 71, 71.

WHE'E'LE'R, C. T., CAMERON, E'. M. & GORI)ON, J. C. (1978). Effects of handling and surgical treatments on nitrogenase activity in root nodules of Alnus glutinosa, with special reference to the appli- cation of indole-acetic acid. Newv Phytol. 80, 175.

WHIEELER, C. T. & LAWRIE', A. C. (1976). Nitrogen fixation in root nodules of alder and pea in relation to the supply of photosynthetic assimilates. In: Symbiotic Nitrogen Fixation in Plants. I.B.P. 7, 497 (LEd. by l'. S. Nutman). Cambridge University P'ress, London.



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Whole Plant Studies on Photosynthesis and Acetylene Reduction in Alnus glutinosa