Regulation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by Carbamylation and 2-Carboxyarabinitol 1-Phosphate in Tobacco: Insights from Studies of Antisense Plants Containing Reduced Amounts of Rubisco Activase^{<a href="#FN1" rid="FN1" class=" fn">1</a>}

Plant Material

Two genotypes of tobacco (Nicotiana tabacum L. cv Wisconsin 38) were used in these experiments: wild type and a type transformed with antisense DNA targeted at the Rubisco activase protein (Mate et al., 1993). The transgenic plants contained T-DNA from pαTACT having antisense genes containing the 3′ two-thirds of the Rca mRNA. The R₁ progeny of the primary transformant A52, which had two T-DNA inserts (Mate et al., 1993), was used. Typically, the leaves of antisense plants had Rubisco activase levels approximately 10% to 20% of those of the wild type. The plants were grown from seed in potting medium consisting of 50% vermiculite, 25% sand, and 25% pine bark in a controlled-environment growth cabinet. They were supplied with one-quarter-strength Hoagland solution every 2 d and exposed to a PPFD of 350 μmol m ^{s with a photoperiod of 12 h. Photoperiod and darkness air temperatures were 25°C and 20°C, respectively.}

Gas Exchange

Gas-exchange measurements with a single-pass system similar to the one described by Mott (1988) were made to determine leaf photosynthetic and respiratory rates. A differential IR analyzer (model 225 Mark 3, Analytical Development Company, Hertfordshire, UK) was used to measure the CO₂ concentration, and a dew-point hygrometer (Dew-10, General Eastern Instruments, Watertown, MA) was used to measure water vapor concentration. A type t thermocouple was used to measure leaf temperature. The upper surface of the leaf was illuminated using a 400-W metal halide lamp. The net CO₂ assimilation rate (A), the stomatal conductance (g_s), and the leaf intercellular CO₂ concentration (c_i) were calculated using the equations of von Caemmerer and Farquhar (1981). Before experiments were initiated the leaves were exposed to a PPFD of 1200 μmol m ^{s for 60 min.}

Typically, the rate of CO₂ assimilation by leaves was measured continually following an increase in PPFD from darkness or 105 to 1200 μmol m ^{s. Before the PPFD was increased, the leaves were exposed to darkness or to the low light intensity for various periods ranging from 10 to 120 min. The RH of the air being supplied to the chamber was 70% during measurements. However, this was increased to 90% during the period of reduced illumination to reduce the decline in} g_s. Gas-exchange data were recorded at 5-s intervals until A had reached a steady state. During the gas-exchange measurements, leaf temperature was 25°C. A was normalized to a c_i of 250 μL L ^{unless otherwise stated, to compensate for changes in the rate of assimilation resulting from changes in} c_i. Normalization assumes that the relationship between A and c_i is linear and passes through the CO₂-compensation point (Woodrow and Mott, 1989).

CA1P Analysis

Leaf discs (1.43 cm^{) were excised from leaves exposed to various light environments and then rapidly frozen in liquid N}₂ within 2 s of excision. The CA1P content was then determined using a method similar to that of Moore et al. (1991). The frozen leaf tissue was ground to a fine powder with a mortar and pestle in liquid N₂ and extracted rapidly with 300 μL of 0.46 n HClO₄. This mixture was centrifuged for 3 min at 13,800g before a 150-μL aliquot of the supernatant was taken and neutralized with 47.5 μL of a solution containing 1.67 n KOH and 0.133 m Hepes. The neutralized extract was incubated at 4°C for 30 min to allow maximal precipitation of KClO₄, and the solution was further clarified by centrifugation for 1 min at 13,800g.

The CA1P concentration in the extracts was determined by measuring its inhibition of purified and carbamylated Rubisco. Rubisco was purified from spinach according to the procedure of Edmondson et al. (1990) and was activated for 1 h at 4°C in a buffer comprising 200 mm Bicine-KOH, pH 8.2, 30 mm NaHCO₃, 40 mm MgCl₂, 5 mm DTT, and 32 μm Rubisco active sites. An equal volume of the CA1P extract was added to the activated Rubisco, making the final concentration 100 mm Bicine-KOH, pH 8.2, 15 mm NaHCO₃, 20 mm MgCl₂, 2.5 mm DTT, and 16 μm Rubisco active sites. The activated Rubisco was incubated with the CA1P extract for 20 min at 25°C before being added to an assay with a final volume of 0.5 mL consisting of 50 mm Bicine-KOH, pH 8.2, 15 mm NaH^CO₃ (specific activity 3.7 GBq mol^{), 20 m}m MgCl₂, 0.5 mm RuBP, and 5 mm DTT. The assay was stopped after 30 s by adding 0.5 mL of 2 n HCl and evaporated to dryness with heating. Acid-stable ^{C fixed by Rubisco was determined using liquid-scintillation counting. Inhibition of Rubisco by CA1P was quantified by comparing with assays containing identical amounts of Rubisco but no CA1P. It was assumed that each inactive Rubisco site was bound with one molecule of CA1P and that all CA1P was bound to Rubisco sites (i.e. negative cooperativity was negligible), because there was always at least a 3-fold excess of Rubisco sites compared with the CA1P concentration.}

Rubisco Activation in the Wild Type

When a wild-type tobacco leaf was exposed to a low light flux (105 μmol m ^{s) for 30 min and then to a sudden increase in PPFD to 1200 μmol m s, there were two kinetically distinct phases during the subsequent increase in} A. The first was a fast phase, presumably representing rapid RuBP production (Woodrow and Mott, 1992), and the second was a slower, exponential phase, representing the production of active Rubisco from inactive forms (Fig. ​(Fig.1A;1A; Woodrow and Mott, 1992). The kinetics of this second Rubisco phase have been used in several studies to examine the activation of Rubisco in leaves of spinach, wild-type tobacco, and anti-activase tobacco (Woodrow and Mott, 1989, 1992; Mott and Woodrow, 1993; Hammond et al., 1998). The same kinetic analysis was used here. We first plotted the ln of the difference between the final A (A_f) and A to confirm that this second phase was linear. It showed exponential kinetics from about 1.2 min on (Fig. ​(Fig.1B).1B). We then used nonlinear regression analysis to fit a curve to the data points in this Rubisco phase. The equation of the curve is:

1

where A_i is the initial extrapolated assimilation rate at time 0 (when the PPFD was increased), k_a is the apparent rate constant for the phase, and t is time after the increase in PPFD. The A_f, A_i, and k_a values for the experiment in Figure ​Figure1A 1A were 20.45 μmol m ^{s, 11.35 μmol m s, and 0.503 min, respectively. The initial velocity of Rubisco activation (}v_i) was calculated by first differentiating Equation 1, which yields the following equation:

2

where A′ is the rate of increase in A. At time 0 this equation becomes

3

where A^_i is the initial rate of increase in A. Using the rate equations and the kinetic constants for carboxylation and oxygenation by Rubisco (von Caemmerer et al., 1994), we converted A^_i into v_i and A_f− A_i into the amount of Rubisco activated during the light-intensity transient. In calculating v_i, we assumed that the RuBP concentration was saturating for Rubisco activity and that respiration did not change (Woodrow and Mott, 1989). For the experiment in Figure ​Figure1A, 1A, v_i was 76.03 nmol active sites m ^{s. We also calculated the proportion of Rubisco active sites that were inactive before the PPFD was increased (}P_r):

4

In the example shown in Figure ​Figure1A,1A, the value of P_r was 0.45.

Figure 1

A comparison of the increase in A upon illumination of a wild-type (A and B) and an antisense tobacco leaf (C and D) following exposure to either darkness or low light flux. The leaf was either illuminated at a PPFD of 105 μmol m ^{s for 30 min (○) or darkened for 60 min (•) before the light flux was increased to 1200 μmol m s at time 0. A,} A plotted over time. B, The ln of the difference between the final assimilation rate (A_f) and the measured A plotted over time. The linear portions of the logged data are exponential and exponential curves were fitted to these portions (solid lines in A and B). C, Same as A except for an antisense plant. D, Same as B except for an antisense plant.

Another experiment was done with the same wild-type leaf that was darkened for 60 min before being exposed to a PPFD of 1200 μmol m ^{s. The kinetics of Rubisco activation were also exponential, but the rate constant was different (Fig.} ​(Fig.1,1, A and B). Analysis of the exponential Rubisco phase revealed that both the P_r (0.66) and the v_i (91.94 nmol sites m ^{s) were higher when the initial condition was darkness instead of low PPFD.}

Novel Phase of Rubisco Activation in Anti-Activase Tobacco

When similar experiments were done using an anti-activase plant, the kinetics of the increase in CO₂ assimilation from low PPFD were slower but still consisted of the two distinct phases present in the wild type (Fig. ​(Fig.1C).1C). The assimilation kinetics from darkness, however, showed a significant qualitative difference from those of the same leaf when the initial PPFD was 105 μmol quanta m ^{s and from those of the wild type (Fig.} ​(Fig.1C).1C). Instead of one slow phase (the Rubisco phase) after the initial fast phase, there were two. The first of these slower phases did not persist beyond 10 min after the increase in PPFD. The second slower phase dominated the time course from about 10 min after the increase in PPFD until a steady state was approached. This phase showed exponential kinetics typical of the Rubisco phase, as indicated by the linearity of a semilogarithmic plot of this portion of the data (Fig. ​(Fig.1D).1D). The curve through the solid points in Figure ​Figure1,1, C and D, is an exponential function (Eq. 1) fitted to the linear portion of the log plot after the novel phase had ceased. From these curve analyses, the A_f values were found to be similar for the two antisense experiments: from darkness (21.79 μmol m ^{s) and from low PPFD (21.58 μmol m s). The} P_r value, however, was higher following 60 min of darkness (0.72) than following 30 min of low PPFD (0.51). The v_i from darkness (12.26 nmol sites m ^{s) was not appreciably different from that from low PPFD (13.75 nmol sites m s).}

To analyze the kinetics of the novel phase we subtracted the contribution of the Rubisco phase from the overall assimilation time course according to the following equation:

5

Therefore, the difference between the two curves (A_d) approaches 0 as t approaches infinity (i.e. steady state). The change in A_d over time was then used to describe the kinetics of the novel phase, which is clearly distinguishable from the fast RuBP phase in the antisense plants (Fig. ​(Fig.2,2, A and B). The latter phase was complete after approximately 2 min. In contrast to the antisense plant, no additional phase could be distinguished in the plot of A_d over time for the wild type (Fig. ​(Fig.2B). 2B).

Figure 2

Resolution of the various phases from the activation of A following illumination of a darkened antisense leaf. A, A trace (○) with an exponential curve fit to the exponential portion (solid line). The amount of Rubisco sites sequestered in the E*R form (A_i) and the v_i were calculated from extrapolation of this exponential curve to time 0. B, Difference between the exponential fit and the measured A (A_d) plotted over time for an antisense plant (○) and a wild-type plant (•). C, Portion of the antisense data after the rapid RuBP-limiting phase and before the later exponential Rubisco phase (when A_d equals 0; see Results) plotted over time. To these data another exponential curve of the same form as that used to characterize the Rubisco phase was fitted (line).

To quantify the contribution of the novel phase to the overall increase in photosynthetic rate, we needed to extrapolate it to 0 time as we had done for the Rubisco phase. Unlike the latter phase, however, the kinetics of the novel phase are apparently more complex. After completion of the RuBP phase (at about 2 min), the velocity (i.e. the rate of change in A_d) decreases gradually with time, and then toward the end, it decreases relatively rapidly to 0 (Fig. ​(Fig.2B).2B). Accordingly, we could not fit the entire novel phase adequately to an exponential function decaying to 0 at infinite time. However, by eliminating the last part of the curve (no more than 10% of the total change in A_d during the phase) and the initial RuBP phase (the first 2.5 min), we could model the kinetics of A_d extremely accurately using an exponential function (Eq. 2). In each case the exponential function was more highly correlated with the data than the linear function. Also, removal of more data at either the beginning or end of the time course had little if any effect on the shape of the fitted exponential curve. An example of the curve fitting is shown in Figure ​Figure2C.2C. In this case, the time course is clearly curved, and in accordance with this, the exponential equation was more correlated with the data than the linear function.

We then used this exponential function to extrapolate A_d to 0 time. This extrapolated value (A_id) was used with the kinetic constants for Rubisco (see above) to calculate both the number and proportion (P_c) of Rubisco sites inactivated by CA1P (i.e. that which accounts for the novel phase) before the PPFD was increased. The latter is given by the following equation:

6

For the example plotted in Figure ​Figure2,2, the number of Rubisco sites in this form was determined to be 4.06 μmol m^{, compared with 10.63 μmol m in the noncarbamylated form. The contribution of} P_c was 0.28, whereas the value of P_r was 0.72. This means that the novel phase accounted for 28% of the total increase in Rubisco activity following the increase in PPFD. Differentiation of the exponential equation fitted to this phase allowed us to determine the initial velocity of Rubisco activation during this phase:

7

where A^_if is the initial rate of increase of this resolved phase and A_fd is the final A_d value determined by the exponential curve-fitting process. This value was negative and had no mechanistic significance. Again, this initial rate of assimilation increase (A^_if) was converted into the initial rate of Rubisco site activation (v_id) using the kinetic constants for Rubisco. The initial velocity of Rubisco activation in the Rubisco phase (v_i) was 12.3 nmol sites m ^{s, whereas} v_id was 15.8 nmol sites m ^s.

Correlation of CA1P Content with the Magnitude of the Novel Gas-Exchange Phase

The next experiments involved testing the hypothesis that the novel phase detectable in the antisense plants reflects removal of CA1P from ECM. We raised this hypothesis because it is known that CA1P regulates Rubisco activity to a degree in darkened tobacco leaves (Servaites et al., 1986; Moore et al., 1991). By varying the dark period before increasing the PPFD, we were able to vary the magnitude of the novel phase (A_fd − A_id) and correlate this with leaf CA1P content. We found that the number of Rubisco sites sequestered in the form responsible for the novel phase (the dark-originating form of inactive Rubisco) indeed correlated linearly with the amount of CA1P extracted from the same leaf after being exposed to the same period of darkness (Fig. ​(Fig.3A).3A). The biochemical assay for CA1P did not exclude the possibility that other naturally occurring, tight-binding inhibitors of the carbamylated Rubisco active site were contributing to the reduced in vitro Rubisco activity that we attributed to CA1P alone. When treated with alkaline phosphatase, however, the inhibitor(s) of Rubisco extracted from darkened tobacco leaves was found to dissociate with kinetics similar to those of purified CA1P (Berry et al., 1987).

Figure 3

A, The correlation of Rubisco sites in the inactive form responsible for the novel gas-exchange phase with leaf CA1P content (r ^{= 0.85). The amount of CA1P and inactive sites was varied by altering the period for which the leaf was darkened. The data are also plotted separately as a function of time in darkness. B, Inactive Rubisco. C, CA1P content (•). CA1P content for the wild-type is also plotted (▵). Inactive Rubisco sites were quantified from gas-exchange measurements made on several leaves of one plant. Leaf CA1P content was determined biochemically using samples taken from one of the leaves used for gas exchange.}

We also used the data presented in Figure ​Figure3A3A to examine the relationship between the time in darkness and the magnitude of the novel phase. We found that this phase had similar sigmoidal kinetics to the CA1P determined biochemically (Fig. ​(Fig.3,3, B compared with C). The formation of CA1P in darkened leaves was also determined for wild-type tobacco using the biochemical assay, and these data show that reduction in the activase concentration had a negligible effect on the rate of CA1P formation and the final steady-state concentration of CA1P (Fig. ​(Fig.33C).

Relationship between CA1P Disappearance and Gas-Exchange Kinetics

Apart from correlating the appearance of the novel phase (which we will now refer to as the CA1P phase) with CA1P concentration, experiments were also conducted to study the relationship between the activation of this form of Rubisco and the metabolism of CA1P in reilluminated leaves in the non-steady state. Following 90 min of darkness, the activation of Rubisco in an anti-activase leaf exposed to a PPFD of 1200 μmol m ^{s was measured using gas exchange, and the kinetics of the CA1P phase were determined. The experiment was repeated with the same leaf, except that instead of making gas-exchange measurements leaf samples were taken at various times and the CA1P content was determined. From the gas-exchange data, the initial amount of inactive Rubisco sites in the dark-originating form was 4.20 μmol m, which correlated with the CA1P content of the leaf (3.57 μmol m; Fig.} ​Fig.4A).4A). During the non-steady state, however, there was less correlation between inactive Rubisco sites and the CA1P content. The leaf CA1P content was higher than the number of inactive Rubisco sites except at low levels of CA1P (Fig. ​(Fig.4B).4B). There was significant deviation from the proportional relationship between CA1P and inactive Rubisco sites that would be expected if CA1P binding was very tight and if CA1P degradation following release from Rubisco was very fast (Fig. ​(Fig.4B). 4B).

Figure 4

A, The relationship between leaf CA1P content determined biochemically (▴) and the dark-originating form of inactive Rubisco sites (○) following an increase in PPFD from darkness to 1200 μmol m ^{s. B, Same data replotted to examine the correlation between leaf CA1P and inactive Rubisco. The dashed line is the relationship expected if CA1P binding to Rubisco sites is complete and CA1P degradation following release is very fast, i.e. a proportional relationship. The amount of CA1P in an antisense leaf predarkened for 90 min was determined for samples taken at various periods after increasing the PPFD to 1200 μmol m s. The activation kinetics of the dark-originating form of inactive Rubisco was previously determined for the same leaf following 90 min of darkness.}