CHANGES WITH AGE IN THE PHOTOSYNTHETIC AND RESPIRATORY COMPONENTS OF THE NET ASSIMILATION RATES OF SUGAR BEET AND WHEAT

CHANGES WITH AGE IN THE PHOTOSYNTHETIC AND RESPIRATORY COMPONENTS OF THE NET ASSIMILATION RATES OF SUGAR BEET AND WHEAT BY D. J. WATSON, J. H. WILSON*, MARGARET A. FORD AND S. A. W. FRENCH Rothamsted Experimental Station, Harpenden, Hertfordshire {Received 9 April 966) SUMMARY The net assimilation rates {E) of sugar beet and wheat growing in a controlled environment, and their components, the rates of photosynthesis {P) and respiration [R) of the whole plant per unit leaf area, were determined at intervals by a method described previously that depends on measuring the decrease in E when photosynthesis is prevented on some days during an experimental period by keeping plants in darkness. An alternative method of changing the duration of photosynthesis, by shortening the daily photoperiod, gave estimates of P and R almost identical with those obtained by shading plants throughout some days. In a period of 50 days from sowing for wheat and 90 days for sugar beet, E decreased by about a half. The value of P was always much greater than R; the smallest ratio of P to i? was 5. Most of the decrease in E with age was caused by decrease in P, and the change in R with age was relatively small. At 30 days after sowing, E of sugar beet was twice that of wheat, wholly because of difference in P. These experiments confirm that, except perhaps in extreme conditions, change in E can safely be attributed to change in photosynthetic rate. INTRODUCTION The net assimilation rate {E) of a plant population the mean rate of increase in total dry weight per unit leaf area during i or 2 weeks represents the excess of the rate of production of dry matter by photosynthesis (P) over the rate of loss by respiration {R) both expressed per unit leaf area. Watson and Hayashi (965) estimated these photosynthetic and respiratory components by measuring the effect on E of preventing photosynthesis during a known fraction of an experimental period. The net assimilation rates of young barley or sugar-beet plants were determined in a controlled environment. In periods of N days {N = 4 or ) during which plants were allowed to photosynthesize on n days distributed evenly over the experimental period and were kept in darkness on the other days, E increased linearly with n over the range 9- days. This was taken to imply that preventing photosynthesis on some days affected neither their rate of photosynthesis on other days nor the rate of respiration of the whole plants per unit leaf area. If so, the slope b of the regression line of on w represents the contribution of i day's photosynthesis to E; the whole photosynthetic component, P, is then given by Nb, and the respiratory eomponent, R, by Nb - E^, where is the net assimilation rate of plants allowed to photosynthesize on all N days. * Present address: School of Agriculture, University of Melbourne, Australia. 500

Changes in net assimilation rate 50 Ef^, P and R are all expressed in the usual units of grams of dry matter per unit leaf area per week. The rates of respiration of sugar-beet and barley plants so determined were similar, so sugar beet had a greater net assimilation rate solely because its rate of photosynthesis was greater. Watson and Hayashi (965) suggested that P and R might alternatively be estimated by measuring the effect on E of varying the daily photoperiod instead of the number of days on which photosynthesis is permitted. They also proposed that the method be used to determine how far change in E with age depends on change in respiration rate per unit leaf area, possibly caused by change in leaf area ratio. Both these suggestions were examined in the work now to be described. METHODS Seven experiments were carried out, three with sugar beet and four with wheat. The environmental conditions were the same for all, except when photoperiod was deliberately varied (Table i). In all experiments the pots were arranged in randomized blocks. Table i. Details of experiments Environmental conditions were as follows. Photoperiod; i6 hours except where indicated otherwise; light intensity: 400 ft-candles, equivalent to 0.08 cal/cm^/min visible radiation, from fluorescent tubes supplemented with tungsten lamps; temperature; in light 20 C, in dark " C; relative humidity; in light 74 %, Sugar beet Wheat m dark 92 Experiment I 2 3 4 5 6 7 Start of experiment (days from sowing) 23 5 78 3 24 39 39 Initial* leaf number 6 9 37 2 9 33 33 Duration of experiment, days (iv) II II T'reatmentst (S), ii(s), 8(S), i5(s),,8, II, 8, 8a, 9, 7, 5,,8, II, 8, I5(U), iob, 5c Initial sample 46 37 8 34 IS 23 23 Replicates Experimental * Including dead and partly expanded leaves. t Eigures denote numbers of full days of photosynthesis (n). 8a in Experiment 3 = days each of 8^ hours, equivalent to 8 days of 6 hours. iob in Experiment 7 = days each of iof hours, equivalent to 0 days of 6 hours. 5c in Experiment 7= days each of 5! hours, equivalent to 5 days of 6 hours. In the sugar-beet experiments (S) denotes small pots; all other treatments were in large pots. All wheat plants were in small pots. (U) in Experiment 7 indicates that the plants were not staked. The sugar-beet plants (var. Klein E) for Experiments -3 were sown on 9 November 964. Plants for Experiment i were grown singly in small pots containing.3 kg of a 4 : I mixture of Rothamsted soil with sand, to which was added.2 g NH4NO3 and.2 g K2HPO4 per pot. Small pots were used because space in the growth rooms was limited and it was judged that pot size would not affect growth before Experiment i ended. Plants for Experiments 2 and 3 were grown singly in larger pots each containing 4.5 kg of the same soil-sand mixture with addition of 3.5 g NH4.NO3 and 3.5 g K2HPO4. per pot. To test whether pot size affected E of older and larger plants, one treatment in Experiment 2 consisted of plants grown in small pots for comparison with the control treatment in large pots. In Experiments 2 and 3, large pots each received 2.3 g 7H2O and small pots 0.75 g, because past experience had shown that sugar-beet 6 7 23

5O2 D. J. WATSON et al. plants grown in the soil used for the experiments were likely to develop magnesium deficiency symptoms in later stages of growth. When necessary, menazon was applied to the soil in solution, to control aphids. The wheat plants (var. Jufy I) for Experiments 4-7 were sown on 2 February 965. They were grown singly in small pots each containing.25 kg of the same soil-sand mixture as in the sugar-beet experiments, with addition of i g NH4NO3 and i g K2HPO4 per pot. In Experiments 6 and 7 (except for one treatment in Experiment 7) the elongating shoots were held erect within strings attached to short stakes round the edges of the pots. Both sugar-beet and wheat seedlings were moved into the controlled-environment rooms immediately after they emerged. The plants required for experiment were selected at random from the whole stock. They were graded by size and allotted to blocks so that plants were as uniform as possible within each block. Treatments were allotted at random within blocks. At the start of each experiment the total leaf area of each plant was obtained by summing estimates of the areas of the individual leaves. The areas of sugar-beet leaves were estimated by rating against a graded series of standards (Williams, 954). The area of the lamina of each wheat leaf was estimated by multiplying the product of its length and breadth by a previously determined factor, and areas of leaf sheaths were estimated by measuring the length and mean diameter of the cylindrical part of each shoot (Watson, Thorne and French, 958). At the same time the initial dry weight of each plant in the experiment was estimated from its leaf area by means of a linear regression coefficient of plant dry weight on leaf area determined by harvesting an initial sample of comparable plants (Watson and Hayashi, 965). In Experiment 3 a regression of plant dry weight on tap root circumference, instead of on leaf area, was used. Plants were shaded individually with inverted metal plant pots or large cardboard cylinders made light-proof and partially covered with aluminium foil sufficient to maintain the temperature under them the same as that of the surrounding air, or, in Experiments 6 and 7, by moving them into a dark-room maintained at the same temperature as the growth-rooms. In choosing the days of darkness for each treatment and the time of day at which shading was applied, the same system was followed as in the experiments of Watson and Hayashi; i.e. the days of darkness were symmetrically disposed about the middle day of the experimental period, different treatments were shaded on different days to minimize the number of plants to be shaded on any one day, and shades were moved only when the lights were on to avoid damage to plants, so each whole day's shading overlapped into two light periods. At the end of each experimental period the plants were harvested and the leaf area and total dry weight of each were determined; E, P and R were then calculated by the methods described by Watson and Hayashi. Leaf area ratio, E (the ratio of leaf area to total dry matter) and R^, the rate of respiration per gram dry weight, were also computed. RESULTS Growth of unshaded plants The growth curves of dry weight and leaf area per plant (Fig. i) are drawn freehand through the mean values for initial and final samples of unshaded plants in each experiment. There are six points for sugar beet, and only four for wheat because two of its

Changes in net assimilation rate 503 final samples also served as initial samples for the following experiments. The dry weight of sugar beet was already greater than that of wheat 20 days after sowing, and the difference increased witb time. The grovfxh rate of sugar beet began to decrease after 80 days. The leaf area of wheat was greater than that of sugar beet at 20 days but increased more slowly, so that after 40 days sugar beet had more leaf area than wheat. Leaf area of sugar beet increased very slowly after 60 days to a maximum at about 80 days. TO 60 50 / / 30 20 io Q. k 30 i 2 i I / 3 i 20 4 i 5 I 6 and i ( // > 0 200 00 ep 80 CT ^ 00 u. 50 I i / \ s K 60 LJ 20 20 60 80 00 0 Days from sov. 20 60 Fig. I. Changes with time in total dry weight (W), leaf area (i), leaf area ratio (F) and net assimilation rate (E) of the unshaded sugar-beet ( ) and wheat ( ) plants. The numbered arrows indicate the mid-points of each experiment. The leaf area ratio {E, Fig. i) of sugar beet was at first very small (less than 50 cm^/g at 23 days after sowing), increased to a maximum of 0 cm^/g after 40 days, and then decreased steadily to 50 cm^/g after 80 days. E of wheat was about 200 cm^/g at 3 and 24 days after sowing, but then decreased to 20 cm'^/g after 50 days. Thus, E was larger for wheat than sugar beet until 50 days after sowing. The mean net assimilation rates of unshaded plants are plotted in Fig. i at the midpoints of the experimental periods. The values of E of both species decreased with time, more rapidly at first than later. Between 30 and 60 days after sowing (mid-points of Experiments i and 2) E of sugar beet was halved, but subsequently it changed very

504 D. J. WATSON e^ a/. little. The value of E of wheat decreased with time throughout the period from 20 to 45 days after sowing. Wheat, like barley, consistently had a smaller E than sugar beet of comparable age; at 30 days after sowing (mid-points of Experiments i and 5) E of sugar beet was double that of wheat, but the difference decreased later. Determination of P and R There are anomalies in the results for sugar beet that must be resolved before P and R can be calculated. In Experiment 2 the treatment means of E for plants grown in large pots (Table 2) seem to show that shading on four days decreased E much less than shading on three extra days (difference between 4 and 7 days of shading). The probable explanation is that the estimate of mean E for unshaded plants was too small. Support for this comes from the independent estimate of E for unshaded plants grown in small pots. Table 2. Treatment means of net assimilation rate (E) in Experiment 2 Large pots Small pots S.E. Days of shading 0 4 7 0 All blocks 38 33 2 46.8 Mean E (g/m^/week) Seven side blocks 29 3 20 46 2.9 Ten central blocks 44 34 2 46 2.4 Table 2 shows that almost all the difference in E between unshaded small and large pots can be attributed to the small E for unshaded large pots in seven blocks at the sides of the growth room; mean E for unshaded large pots in these blocks was obviously anomalous because it was less than E for pots given 4 days shading. Mean E for unshaded large pots in the remaining ten blocks in the centre of the room was much larger and nearly the same as mean E for unshaded small pots, which had identical values for central and side blocks. The explanation apparently depends on a difference in growth habit between shaded and unshaded plants; the shaded plants had conspicuously more erect leaves than the unshaded ones presumably because constriction within the cylindrical shades for part of the time had a persistent effect on the posture of the petioles. They were therefore neither shaded by neighbouring plants, nor did they shade their neighbours. In contrast the outer leaves of unshaded plants in large pots were more prostrate and so liable to be overshadowed by their neighbours. This apparently occurred in the side blocks where they were overshadowed by an outer row of plants (not in the experiment), raised closer to the lights to compensate for smaller light intensity towards the sides of the room. Unshaded plants in small pots were not so affected, probably because they were raised on wooden blocks to offset the smaller pot size. The results for unshaded plants in large pots were therefore rejected, and mean E for unshaded plants in small pots was used instead in fitting the regression of.e on M (Fig. 2). In Experiment 3, the plants were spaced more widely to prevent mutual shading, but the results plotted in Fig. 2 suggest that it was not entirely avoided as mean E for unshaded plants again appeared anomalously small. As the effect on E in Experiment 3, of shading plants each day for seven-fifteenths of the standard photoperiod, did not differ significantly from that obtained by covering plants for 7 out of days, the means

Changes in net assimilation rate 5 5 for both these treatments were included in calculating the regression of E on days of photosynthesis. No evidence of interference between neighbouring pots of different treatments was found in the wheat experiments. The supports holding plants erect in Experiments 6 and 7, had no effect on E\ mean values of E in Experiment 7 for staked and unstaked unshaded pots were nearly the same (37.7 and 38.+0,83 g/m^/week, respectively), and both were included in the analysis of the results. Fig, 2 Fig, 3 Figs, 2 and 3, Linear regression lines of net assimilation rate {E) on number of days of photosynthesis in the experimental period, fitted to the values for sugar beet in Experiments -3 (Fig, 2), and for wheat in Experiments 4-7 (Fig. 3), The open triangles indicate values obtained in Experiments 3 and 7(i ^)by shortening the daily photoperiod, instead of shading plants (see text). Vertical lines represent least significant differences between values within an experiment {P = 0,05), Figs. 2 and 3 show that when the treatment means of E were plotted against number of days of photosynthesis during the experimental period of each experiment, the points lay close to straight lines, and the deviations from straight lines were never statistically significant. Eor Experiment 7, the mean values of E for plants shaded every day for onethird or two-thirds of the normal photoperiod were plotted in Fig, 3 as though they were shaded for 5 or 0 whole days respectively during the experimental period of days. The deviations of the points for Experiment 4 from the straight line in Fig. 3 suggest

5o6 D. J. WATSON et al. that there was slight curvature in the relation of mean E to number of days of photosynthesis, but when a quadratic regression was fitted to the points (Fig. 3), the variance attributable to the second order term was not significantly greater than the error variance, as the following analysis shows. d.f. Variance Linear regression i 4989.8 Additional for quadratic regression I 7-4* Deviations I 0.4 Error 40 20.4 * Variance ratio = 3.45; 5 % Probability level = 4.08. The values of b, the linear regression coefficient of E on n, and of E and n, the means over all treatments, were calculated for each experiment, and from them E'^, the value of E on the regression line when n = N, which is the best estimate of the mean net assimilation rate of unshaded plants, its components P and R and their standard errors, were computed as described by Watson and Hayashi (Table 3). The magnitude of R is shown in the figures by extrapolating the regression lines to give the values for E for zero days of photosynthesis {n = o). Table 3. Calculation of P and R components of E b Ea N ^^(g/m^/week) P R PIR F (cm^/g) Expt I 6.4 + 0.23 7o.9±o.84-3 IS 94 ±.2 96 ±3=i i.6±2.8 60.0 96 Sugar beet Expt 2 33-3±i-o6 II-3 47 ±-7 54 ±56 7-i±4-4 7-5 III 79 Expt 3 3-4±o-4i 28.6±i.i7 44 ±2.2 5 ±6. 6.9 ±4.4 7-3 47 32 Expt 4 7. ±0.26 42. ±0.58 8 II 63 ± I.O 78 ±2.9 4.3 ±2.2 5-4 202 290 Wheat Expt 5 Expt 6 3-3±o-ii 34.5 ±0.32.3 46 ±0.5 49 ±-7 8.2 98 53 2.7±O. II-3 37 ±o-6 40 +2. 0 3-2 ± I. 6 2.5 5 50 4 Expt 7 2.7±O.O 27.5 ±0.4.25 38 ±0.6 4 ±-5 3-4±i-i 2. 3 52 The values of R for sugar beet in Experiments 2 and 3 were nearly the same; that for Experiment i, though smaller, was not significantly different. It seems unlikely that the youngest plants could have such a small respiration rate, and a reasonable interpretation of the results is that R changed little with time, so that the decrease in E with age between Experiments i and 2 was almost wholly the result of decrease in P. Neither E nor its components differed significantly between Experiments 2 and 3. The experiments on wheat were more precise than those on sugar beet. The young wheat plants of Experiment 4 had larger P and much larger R, and hence a smaller ratio of P to R, than the older plants in later experiments. Consequently, the change in P exceeded that in-between Experiments 4 and 5, and equalled it between Experiments 5 and 6; decrease in P with age was sufficient to account for the decrease in E. The different methods of varying the length of time for which photosynthesis was permitted during the experimental period, compared in Experiments 6 and 7, gave nearly identical results (Fig. 3 and Table 3).

Changes in net assimilation rate 507 DISCUSSION The method of estimating P and R is valid only if shading plants on some days changes neither their rate of respiration nor their rate of photosynthesis on the remaining days when they are illuminated. When both these requirements are satisfied, E will be linearly related to the number of days of photosynthesis during the experimental period. No significant curvature in this relation was found in any of the experiments, but in Experiment 4 with young wheat plants successive increases in the number of days of shading (decreases in n) apparently caused less than proportionate decreases in E. If this apparent curvature were real, the explanation might be that shading on some days increased the rate of photosynthesis on the other days through more complete removal of photosynthetic products from the leaves, or decreased the rate of respiration through depletion of substrate. If shading has either of these effects P and R will be underestimated when calculated from the linear regression of E on n. If it changes the respiration rate, P and R cannot be estimated with certainty, but if it affects only the rate of photosynthesis and the respiration rate remains independent of n, the slope of the quadratic regression line when n = N instead of the linear regression coefficient b should give correct though less precise estimates of P and R. When so calculated for Experiment 3, the values of P and R were 96 + 0.0 and 3+9.3 g/m^/week, respectively, instead of 78+ 2.9 and 4 + 2.2 (Table 3), when calculated from b. Tbe differences between the two methods of calculation were not significant, because of the large standard errors of the estimates from the quadratic curve, and the conclusions about the effects of age on E, P and R were unaffected. Watson and Hayashi noted that the most efficient experimental arrangement would consist of only two treatments comparing unshaded plants with plants shaded for the maximum possible number of days in the range within which the relation oi E to n can be taken as linear. With older plants, as in Experiment 6, the number of days of shading can safely be up to half of the days in the experimental period, but the results of Experiment 4 suggest that, witb very young plants, the number of days of shading may not be more tban 2 or 3 if bias in the estimates of P and R is to be avoided. The anomalous results of Experiment 2 are a warning of the need to avoid too close spacing of plants. The nearly identical results of Experiments 6 and 7 (Table 3) and the good fit of the linear regressions of on M in both experiments (Fig. 3) justify the assumptions on which the method of calculating P and R depends, viz. that shading, whether for whole days or for an additional part of each day, changes E merely by shortening the duration of photosynthesis during the experimental period and does not affect the rates of photosynthesis or respiration. Varying the photoperiod gave slightly more accurate estimates of P and R than shading for whole days; it may often be the more convenient procedure, and perhaps the preferable one, especially for very young plants, if as Experiment 7 suggests the relation of E to photoperiod is linear over a very wide range. The net assimilation rate {Efi) of sugar-beet plants was considerably greater than that of wheat plants of the same age and size (Fig. i), in agreement with previous comparisons of sugar beet and cereals (Watson, 947; Thorne, i960; Watson and Hayashi, 965). The larger E of sugar beet was the result of larger P, not smaller R. At about 30 days after sowing both sugar beet and wheat had similar respiration rates (Table 3, Experiments i and 5) but P of sugar beet was twice that of wheat, as Watson and Hayashi (965) found.

D. J. WATSON e^ a/. Table 3 shows clearly that decrease with age in E of both sugar beet and wheat was mainly the result of decrease in the rate of photosynthesis {P). The rate of respiration {R) did not increase with time except possibly during the early growth of sugar beet. How much of the decrease in P was from increased self-shading as leaf area increased, and how much from change in internal factors is not known. In young wheat plants both P and R decreased with age, so the change in E was less than in P (Table 3). The decrease with age in leaf area ratio {E, Fig. i) evidently played little part in determining the change in E. Thus, the decrease in F of sugar beet by more than half between Experiments 2 and 3 (Table 3) when the storage root was expanding rapidly was not accompanied by an increase in R; apparently it was offset by a smaller respiration rate per gram dry weight {Rw) so that R remained unchanged. In all the experiments P greatly exceeded R; the smallest ratio of P to i? was 5 (Table 3, Experiment 4). The conditions in the growth rooms, with long photoperiod, high temperature in the light and lower temperature in the dark, and constant moderate light intensity throughout the photoperiod, favoured a large daily photosynthetic output, and in such circumstances only very wide variation in respiration rate could have an appreciable effect on E. In less favourable field conditions respiratory loss may become a more important determinant of net assimilation, but in general it is evidently safe to interpret change in Z? as a measure of change in photosynthetic rate. ACKNOWLEDGMENTS We thank Dr. G. N. Thorne for helpful advice and criticism and other members of the Botany Department who gave technical assistance. REFERENCES THORNE, G. N. (i960). Variations with age in net assimilation rate and other growth attributes of sugar beet, potato and barley in a controlled environment. Ann. Bot., N.s., 24, 356. WATSON, E). J. (947). Comparative physiological studies on the growth of field crops. I. Variation in net assimilation rate and leaf area between species and varieties and within and between years. Ann. Bot., N'.S., II, 4. W.-^TSON, D. J. & HAYASHI, K. (965). Photosynthetic and respiratory components of the net assimilation rates of sugar beet and barley. New PhytoL, 64, 38. WATSON, D. J., THORNE, G. N. & ERENCH, S. A. W. (958). Physiological causes of differences in grain yield between varieties of barley. Ann. Bot., N.s., 22, 32. WILLIAMS, R. E. (954). Estimation of leaf area for agronomic and plant physiological studies. Aust. J. agric. Res., 5, 235.