Jul 30, 1984 - conclude that photosynthesis could be limited by the concentra- tion of either of the two substrates for the reaction, CO2 and. ' Supported by ...
Plant Physiol. (1984) 76, 968-971 0032-0889/84/76/0968/04/$0 1.00/0
Photosynthesis and Ribulose 1,5-Bisphosphate Concentrations in Intact Leaves of Xanthium strumarium L.' Received for publication May 30, 1984 and in revised form July 30, 1984
KEITH A. MoTT2, RICHARD G. JENSEN*, JAMES W. O'LEARY, AND JOSEPH A. BERRY Departments of Biochemistry and Plant Sciences, University ofArizona, Tucson, Arizona 85721 (K.A.M., R.G.J.); Environmental Research Laboratory, Tucson International Airport, Tucson, Arizona 85706 (J.W.O'L.); and Carnegie Institution of Washington, 290 Panama Street, Stanford, California 94305
(J.A.B.) ABSTRACT The interacting effects of the rate of ribulose 1,5-bisphosphate (RuBP) regeneration and the rate of RuBP utilization as influenced by the amount and activation of RuBP carboxylase on photosynthesis and RuBP concentrations were resolved in experiments which examined the kinetics of the response of photosynthesis and RuBP concentrations after step changes from a rate-saturating to a rate-limiting light intensity in Xanthium strumariumn. Because RuBP carboxylase requires several minutes to deactivate in vivo, it was possible to observe the effect of reducing the rate of RuBP regeneration on the RuBP concentration at constant enzyme activation state by sampling very soon after reducing the light intensity. Samples taken over longer time periods showed the effect of changes in enzyme activation at constant RuBP regeneration ra* on RuBP concentration and photosynthetic rate. Within 15 s of lowering the light intensity from 1500 to 600 microEinsteins per square meter per second the RuBP concentration in the leaves dropped below the enzyme active site concentration, indicating that RuBP regeneration rate was limiting for photosynthesis. After longer intervals of time, the RuBP concentration in the leaf increased as the RuBP carboxylase assumed a new steady state activation level. No change in the rate of photosynthesis was observed during the interval that RuBP concentration increased. It is concluded that the rate of photosynthesis at the lower light intensity was limited by the rate of RuBP regeneration and that parallel changes in the activation of RuBP carboxylase occurred such that concentrations of RuBP at steady state were not altered by changes in light intensity.
The identification of factors limiting the rate of photosynthetic CO2 fixation under various environmental conditions is of considerable importance for a wide range of plant studies. Since the major rate-determining step in this process is the carboxylation of RuBP3 by the enzyme RuBP carboxylase, it is reasonable to conclude that photosynthesis could be limited by the concentration of either of the two substrates for the reaction, CO2 and
RuBP, or by the activity of RuBP carboxylase present in the tissue. It has been demonstrated that the carboxylase is activated by CO2 and Mge2 in vitro (6), and that the level of activation does vary in vivo (8). Therefore, the activity of RuBP carboxylase present will be influenced by both the amount of enzyme and the degree to which the enzyme is activated. Although CO2 limitation of photosynthesis is easily determined by measuring photosynthetic response to CO2 concentrations, the roles of RuBP concentration and the amount and activation state of RuBP carboxylase are not clear. The kinetic analysis of RuBP carboxylase of Farquhar (3) provides a basis for experiments to determine whether the rate of CO2 uptake in photosynthesis under different environmental conditions is limited by the capacity to regenerate RuBP or by the capacity of RuBP carboxylase to use RuBP. It was proposed that the concentration of RuBP should be rate-saturating if photosynthesis was limited by the capacity of the carboxylase to use RuBP, and rate-limiting if photosynthesis was limited by RuBP regeneration rate. The analysis takes into account the observation that the concentration of active sites of RuBP carboxylase in the stroma of an intact chloroplast is very high relative to the Km of these sites for RuBP. Because ofthis, at one CO2 concentration and one activation state of RuBP carboxylase, RuBP concentration and RuBP consumption should be linearly related for RuBP concentrations which are below the concentration of active sites. For RuBP concentrations above binding site concentration, RuBP consumption rate should be saturated with respect to RuBP. Unfortunately, the measurements of photosynthesis and RuBP concentration lead to contradictory interpretations according to this theory. In the green algae Chlamydomonous reinhardtii and in spinach leaf cells, RuBP concentrations were found to be below binding site concentration at rate-limiting light intensities and at rate-saturating light intensities if CO2 concentrations were high (2, 10), indicating that RuBP supply was limiting under these conditions. In intact leaves, however, RuBP concentrations were found to be saturating at rate-limiting light intensities in two separate studies (1, 8), which would indicate that RuBP regeneration did not limit photosynthesis at rate-limiting light intensities. From the studies of Perchorowicz et al. (8) and Perchorowicz and Jensen (9) it is clear that RuBP carboxylase activation state can change in response to light intensity. The predicted relationship between RuBP consumption and RuBP concentration should be more complex if the carboxylase activation state changes, possibly making it more difficult to resolve the roles of RuBP regeneration rate and the amount and activation state of the carboxylase as rate-determining factors. To clarify the relative roles of RuBP regeneration rate and activation of RuBP carbox-
' Supported by The Science and Education Administration of the United States Department of Agriculture under grant 82-CRSR-l-1010, and by grants PCM 82-07687 and DEB 8110202 from the National Science Foundation. This is Arizona Agricultural Experiment Station Paper No. 3927 and CIW-DPB publication No. 865. 2Current address: Carnegie Institution of Washington, Department of Plant Biology, 290 Panama St., Stanford, CA 94305. 3 Abbreviations: RuBP, ribulose 1,5-bisphosphate; C,, intercellular CO2 concentration. 968
PHOTOSYNTHESIS AND RuBP CONCENTRATIONS ylase, it is necessary to observe the effects of changes in one while the other remains constant. To do this we have taken advantage of the observation of Perchorowicz et aL. (8) that RuBP carboxylase required several minutes or more to deactivate following a step change from high to low light intensity. During the time immediately following the drop in light intensity they found that RuBP concentrations were low and possibly limiting, but rose gradually to the steady state values after several minutes at the reduced light intensity. This observation formed the basis of the approach used in this study with Xanthium strumarium. The experiments reported here were designed to determine if RuBP regeneration rate limits photosynthetic rate at subsaturating light intensities if the carboxylase activation state remains unchanged, and to determine to what extent RuBP regeneration rate and RuBP carboxylase activation limit photosynthesis at steady state.
MATERIALS AND METHODS Cocklebur (Xanthium strumarium L.) seeds were collected from a perennially wet canyon at the base of the Santa Catalina mountains near Tucson, Arizona. Seeds were soaked 3 d in tap water, germinated in vermiculite, and potted when seedlings emerged. Plants were grown in a controlled environment growth room with a light intensity of approximately 450 suE m-2 * s-' at the top of the plants. Photoperiod was 14 h with day and night temperatures of 30 and 20°C, respectively. Plants were watered as necessary, and 500 ml of half-strength modified Hoagland solution was applied at each watering. Leaves used for study were fully expanded with no more than two fully expanded leaves above them, and were selected for uniformity of appearance. For gas exchange measurements, leaves were inserted between two aluminum blocks with circular chambers 2.5 cm in diameter above and below the leaf. The top of the upper chamber and the bottom of the lower chamber were constructed of parafilm. Mounted in the top aluminum block directly above the chamber was an aluminum tube with an inside diameter of 2.5 cm, which served as a guide tube for a stainless steel cutting tube which had an outside diameter of 2.5 cm. When the appropriate gas exchange conditions existed in the chamber, the cutting tube was pushed quickly down through the entire chamber, cleanly slicing out the circular area of leaf as well as the top and bottom of the chamber and plunging them into a container of liquid nitrogen held immediately below the chamber. It was estimated that the leaf tissue was killed within 0.5 s. Light was provided by two 500-w tungsten lamps and delivered to the chamber by two fiber optic bundles which were mounted in the guide tube. Light intensity at the level of the leaf was measured using a Li-Cor LI- 170 quantum meter and was attenuated with neutral density screens between the lamps and fiber optic bundles. CO2 uptake and water loss by the leaf were determined with a gas exchange system capable of measuring these parameters for each side of the leaf independently and simultaneously (for details, see Ref. 7). All measurements were made at 21% 02 with leaf temperature kept at 25C and water vapor pressure difference between the leaf and the chamber air kept at 15 mbar. Photosynthesis, transpiration, stomatal conductance, and internal CO2 concentration were determined using the equations given by von Caemmerer and Farquhar (1 1). Following the rapid freezing of the leaf disc, RuBP levels were measured using the technique of Perchorowicz et al. (8) except that the samples were extracted in 5% HCl04 rather than acetone. Chl per unit area was determined by extraction of similar leaves with methanol or 80% (v/v) acetone, and was found to be very constant among leaves. RuBP carboxylase enzyme active site concentration was not measured directly in these experiments because of difficulty in extracting and activating the enzyme. However, assuming a Km -
969
CO2 of 330 y1lI`' and a kIc,, of 2.5 s-', we estimate that a binding site concentration (for RuBP to the enzyme catalytic sites) of 100 to 120 nmol-mg-' Chl would be required to account for the rates of photosynthesis observed. This binding site concentration is similar to values reported for spinach (5, 12). RESULTS AND DISCUSSION Before attempting non-steady state experiments designed to isolate the effects of RuBP regeneration rate and RuBP carboxylase activation state on the rate of photosynthesis, it was necessary to first determine steady state values for photosynthesis and RuBP concentrations under different environmental conditions. Figure 1 shows the steady state response of photosynthesis and RuBP concentration to intercellular CO2 concentration (C1) at light intensities of 1500 and 600 gE m-2_s-'. In this study, photosynthetic rates increased dramatically with Ci for values below 280 ,l.l'-, but were relatively insensitive to Cs for values over 280 M1l 1-'. von Caemmerer and Farquhar (11) postulated that this type of response was due to carboxylase limitation of photosynthesis at low Cs and RuBP regeneration rate limitation at high Cp. Although the general form of the photosynthetic response to Cs was similar at the two light intensities, photosynthetic rates were lower at 600 gE.m 2s-' than at 1500 MEEmM2.s-' for any given C*. Since all other conditions were identical, it follows that the reduced rate of photosynthesis was the result of limiting light intensities. Farquhar et al. (4) postulated that limiting light intensities should be characterized by limiting RuBP regeneration rates, causing RuBP concentration to be below binding site concentration. However, RuBP concentrations as a function of Ci at 600 MEm-2s-' were not different than those at 1500 uEm2~'. These RuBP concentrations were above 120 nmolmg-' Chl and presumably saturating at C, less than 280 jl.I- 1,
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INTERCELLULAR p(CO2), C j1(I)M FIG. 1. Effect of C~on photosynthetic rate and RuBP concentration at 1500 (@) or 60 (O) ;Em2 s .
970 MOTT but photosynthesis was lower at the lower light intensity. This is consistent with lower steady state activation ofRuBP carboxylase at the lower light intensity and, on this basis alone, the rate appeared to be limited by the activation state. This is in agreement with the results of Perchorowicz et al. (8). For Ci above 280 gl*l' RuBP concentrations were fairly constant at 100 to 120 nmol mg-' Chl which is close to the estimated binding site concentration. This low RuBP concentration plus the shallow rise in photosynthesis with increases in C, above 280 gl- 1-I suggest that RuBP regeneration rate is limiting photosynthesis in this region. However, RuBP concentrations do not appear to decline sufficiently to be strictly compatible with theory. This is in agreement with the results of Badger et al. (1). To examine the effect of lower light intensity and presumably lower rates of RuBP regeneration on RuBP concentration independent of a change in carboxylase activation state, RuBP concentrations were measured at various times after a step change from saturating to subsaturating light intensity. In these experiments, the leaf was allowed to Equilibrate for at least 1 h at 1500 ;E-m-2s- ' and 110 ,ul-I' Ci. At this C, steady state, RuBP concentrations were approximately 200 nmol . mg-' Chl for both 1500 and 600 MAE a m-2.s-' (Fig. 1). However, RuBP concentrations measured immediately following a drop in light intensity from 1500 to 600 tE. m21 s-' fell to approximately 110 nmol. mg7' Chl (Fig. 2). This is well below the steady state values at that light intensity and C,, and is also slightly below the estimated binding site concentration of 120 nmol-mg-' Chl. RuBP concentrations were fairly constant at this low value for the first 3 min after lowering the light intensity, but did rise with longer times.(Fig. 2). By 60 min after the change in light intensity, RuBP concentrations had risen to the steady state value (200 nmol.mg-' Chl observed at that light intensity and C, (Fig. 1). This slow rise in RuBP concentrations was presumably due to deactivation of RuBP carboxylase as previously demonstrated using wheat seedlings (8). If the activation state of RuBP carboxylase changes more slowly than the rate of RuBP regeneration immediately following a drop in light intensity, then it should be possible to demonstrate the linear relationship between RuBP concentration and photosynthesis predicted by Farquhar (3). To test this, leaves were equilibrated at 1500,gE * m2 . s-' and light intensity was dropped to 600, 400,or 180 sE.m-2s'. RuBP concentrations sampled during the first 3 min following the change in light intensity (Fig. 2) are plotted versus the corresponding photosynthetic rates (Fig. 3). The RuBP concentrations were 80 and 40 nmol-mg-' Chl -
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2 3 60 15 30 45 MINUTES AFTER CHANGE FROM I500JJE(m2.s)' FIG. 2. RuBP concentrations after changing light intensity from 1500 ME m2 s-' to 600 (0), 400 (0), or 180 (A)gE * m.2 * s-'.
Plant Physiol. Vol. 76, 1984
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