The Regulatory Properties of Rubisco Activase Differ among Species and Affect Photosynthetic Induction during Light Transitions

Rubisco ’ s catalytic chaperone, Rubisco activase (Rca), uses the energy from ATP hydrolysis to restore catalytic competence to Rubisco. In Arabidopsis ( Arabidopsis thaliana ), inhibition of Rca activity by ADP is ﬁ ne tuned by redox regulation of the a -isoform. To elucidate the mechanism for Rca regulation in species containing only the redox-insensitive b -isoform, the response of activity to ADP was characterized for different Rca forms. When assayed in leaf extracts, Rubisco activation was signi ﬁ cantly inhibited by physiological ratios of ADP to ATP in species containing both a -Rca and b -Rca (Arabidopsis and camelina [ Camelina sativa ]) or just the b -Rca (tobacco [ Nicotiana tabacum ]). However, Rca activity was insensitive to ADP inhibition in an Arabidopsis transformant, rwt43, which expresses only Arabidopsis b -Rca, although not in a transformant of Arabidopsis that expresses a tobacco-like b -Rca. ATP hydrolysis by recombinant Arabidopsis b -Rca was much less sensitive to inhibition by ADP than recombinant tobacco b -Rca. Mutation of 17 amino acids in the tobacco b -Rca to the corresponding Arabidopsis residues reduced ADP sensitivity. In planta, Rubisco deactivated at low irradiance except in the Arabidopsis rwt43 transformant containing an ADP-insensitive Rca. Induction of CO 2 assimilation after transition from low to high irradiance was much more rapid in the rwt43 transformant compared with plants containing ADP-sensitive Rca forms. The faster rate of photosynthetic induction and a greater enhancement of growth under a ﬂ uctuating light regime by the rwt43 transformant compared with wild-type Arabidopsis suggests that manipulation of Rca regulation might provide a strategy for enhancing photosynthetic performance in certain or an ATP-regenerating system consisting of 5 M phosphocreatine units 2 1 phosphocreatine M ADP ATP at a ratio of 0.11 or 0.33 in the absence of ATP-regenerating system. Measurements of ADP concentration at the end of the showed that the ratio of ADP to ATP and the total adenine nucleotide not change markedly during the assay. The rate of Rca activity was previously Salvucci,

The activity of Rubisco, the enzyme that catalyzes CO 2 assimilation in photosynthesis, is regulated by Rubisco activase (Rca), a specific catalytic chaperone (Spreitzer and Salvucci, 2002;Portis, 2003). Like other AAA+ ATPases (Snider et al., 2008), Rca uses the energy from ATP hydrolysis to remodel the conformation of its target protein, Rubisco. The conformational changes induced by Rca restore catalytic competence to Rubisco active sites that have been inactivated by the unproductive binding of sugar phosphates, including the substrate ribulose 1,5-bisphosphate (RuBP; Wang and Portis, 1992). Because of the requirement for ATP hydrolysis and the inhibition of activity by ADP Portis, 1988, 1989), Rca adjusts the rate of CO 2 fixation to the rates of electron transport activity via changes in the activation state of Rubisco (Salvucci et al., 1985). As a result of this coordinate regulation, the light response of Rubisco activation closely resembles the light response of CO 2 assimilation, and the levels of RuBP under steady-state conditions are relatively constant over a wide range of irradiance levels (Perchorowicz et al., 1981;Dietz and Heber, 1984).
Many plant species express two isoforms of Rca, a and b, that are both active in ATP hydrolysis and Rubisco activation (Shen et al., 1991;Salvucci et al., 2003). In some plant species, these isoforms are the products of an alternative splicing event that generates two polypeptides, which are identical except for a 20to 30-amino acid extension at the C terminus of the longer a-isoform (Werneke et al., 1989). In cotton (Gossypium hirsutum), soybean (Glycine max), and presumably other plant species, separate genes encode the two isoforms of Rca (Salvucci et al., 2003;Yin et al., 2010). In these species, the amino acid sequences of the overlapping regions of the aand b-polypeptides are very similar, and the C-terminal extension of the longer a-isoform is similar to the extension produced by alternative splicing (Supplemental Fig. S1).
Our current understanding of the role of the two Rca isoforms is based primarily on investigations with Arabidopsis (Arabidopsis thaliana; Zhang and Portis, 1999;Zhang et al., 2001;Wang and Portis, 2006). The C-terminal extension of the a-Rca contains two redox-regulated Cys residues that are modulated by thioredoxin f (Zhang and Portis, 1999). When these residues are oxidized to a disulfide, the affinity for ATP decreases and enzyme activity is more sensitive to inhibition by ADP. Physiological ratios of ADP to ATP significantly inhibit the activity of the Arabidopsis a-Rca when in the oxidized state, but inhibition is much less when this isoform has been reduced by thioredoxin. In contrast, the shorter Arabidopsis b-Rca is not redox regulated and is less sensitive to inhibition by ADP (Zhang and Portis, 1999). Mixing experiments with recombinant Rca have shown that the properties of a-Rca are conferred to the heterooligomer, providing a mechanism for redox regulating the Rca holoenzyme (Zhang et al., 2001). In this way, Rca is similar to the chloroplastic glyceraldehyde 3-P dehydrogenase (GAPDH), which also has both redox-regulated (GAP-B) and non-redox-regulated (GAP-A) forms that differ by a C-terminal extension (Baalmann et al., 1996). Like Rca (Zhang and Portis, 1999;Zhang et al., 2001), redox regulation of two Cys residues in the extension exerts master control over the mixed GAPDH oligomer.
Some plant species, including members of the Solanaceae family, as well as maize (Zea mays) and green algae, express only the shorter b-Rca . The b-Rca in these species is not responsive to redox regulation, even though the activation state of Rubisco in these plants is modulated by irradiance (Salvucci and Anderson, 1987) and seems to be associated with the redox status of the chloroplast (Ruuska et al., 2000). With GAPDH, all higher plants appear to have both chloroplastic isoforms, but the non-redoxsensitive form, Gap-A, can be regulated indirectly by thioredoxin through the binding of the small chloroplast protein CP12 (Trost et al., 2006). By analogy, a similar association with CP12 or a CP12-like protein could provide a means of conferring redox sensitivity to b-Rca in species that have only this Rca isoform. However, no association of Rca was observed when the native CP12 complex and other high-molecularmass species were isolated from tobacco (Nicotiana tabacum) chloroplasts (Carmo-Silva et al., 2011b).
In this study, the regulation of b-Rca activity was examined both in vivo and in vitro in plant species that contain only one (i.e. b-) or both (a-and b-) Rca isoforms. The response of enzyme activity to physiological ratios of ADP to ATP was measured for the native Rca in leaf extracts, as well as for recombinant Arabidopsis and tobacco enzymes, to determine the sensitivity of b-Rca to ADP in different species. In addition, experiments were conducted with transgenic Arabidopsis plants containing variants of b-Rca to determine the link between Rca regulation and photosynthetic induction. The results suggest a new strategy for enhancing photosynthetic performance under variable light environments based on altering the regulatory properties of Rca to increase the rate of photosynthetic induction.

ADP Sensitivity of Rubisco Activation by Rca in Leaf Extracts
The sensitivity of Rca activity to inhibition by ADP was determined by measuring the rate of Rubisco activation in leaf extracts in the presence of physiological ratios of ADP to ATP (Fig. 1). The rates of Rubisco activation were inhibited by increasing ratios of ADP to ATP in Arabidopsis and camelina (Camelina sativa), two species that express both the a-Rca and b-Rca isoforms, and in tobacco, a species that expresses only Figure 1. Response of Rca activity to ADP in plants with different Rca forms. The rate of Rubisco activation by Rca in leaf extracts was measured at 30˚C in the presence of either 5 mM ATP plus an ATP-regenerating system (0.00 ADP/ATP) or a ratio of 0.11 or 0.33 ADP/ATP (total nucleotide concentration of 5 mM). The Arabidopsis transgenic line rwt43 expresses only the Arabidopsis b-Rca and the line Tob-DAt (S2) expresses a chimeric b-Rca that resembles the tobacco protein but has the Rubisco recognition domain from Arabidopsis. Rates of Rubisco activation are expressed as v i /v c (the inhibited velocity/control velocity, i.e. the rates determined in the presence of ADP relative to the control rate with ATP alone). Control rates were 0.17 6 0.02, 0.20 6 0.00, 0.04 6 0.01, 0.10 6 0.01, and 0.13 6 0.01 Rubisco sites activated per min for Arabidopsis wild type, rwt43, Tob-DAt (S2) , tobacco, and camelina, respectively. Values are means 6 SE of four to six samples. Different letters at the base of each bar denote significant differences (P , 0.05) between the rates at each ratio of ADP to ATP. b-Rca. A similar response was observed in the Arabidopsis line Tob-DAt (S2) , a transformant of the rca null mutant that expresses a chimeric b-Rca, which is identical to tobacco Rca except for inclusion of the Rubisco recognition domain from Arabidopsis (Kumar et al., 2009). Compared with the control rate measured in the absence of ADP, the rates of Rubisco activation in these plants were inhibited by 40% to 60% at 0.11 ADP/ATP and by 70% to 80% at 0.33 ADP/ATP (Fig.  1). In contrast, the activity of Rca in leaf extracts of the Arabidopsis line rwt43, a transformant of the rca null mutant that expresses only the Arabidopsis b-Rca (Zhang et al., 2002), was not inhibited by these ratios of ADP to ATP (P . 0.05).

Modulation of Rubisco Activation by Irradiance in Plants with Different Rca Forms
To relate differences in ADP sensitivity to the in planta activity of Rca, the activation state of Rubisco was measured in plants exposed to high irradiance and after transfer to low irradiance ( Table I). The activation state of Rubisco decreased when plants were transferred from high to low irradiance in wild-type Arabidopsis, tobacco, and camelina and in the Arabidopsis Tob-DAt (S2) transformant. In contrast, there was no significant difference (P . 0.05) in Rubisco activation state at high and low irradiance in the Arabidopsis rwt43 transformant. Lower initial Rubisco activities accompanied the reduced Rubisco activation states at low irradiance, whereas Rubisco total activities were not significantly affected (P . 0.05) by irradiance level, except in tobacco (Table I). In all likelihood, the decrease in total Rubisco activity in tobacco was caused by 2-carboxyarabinitol-1-P, a naturally occurring tightbinding inhibitor of Rubisco that accumulates in the dark and at low light in many plant species, including tobacco (Servaites et al., 1986), but not in Arabidopsis and other plant species (Salvucci et al., 1986).
In species that do not accumulate 2-carboxyarabinitol-1-P, the activation state of Rubisco in the dark, when the RuBP concentration is low, is dependent on the concentration of CO 2 and can be greater than at low irradiance (Perchorowicz et al., 1981;Salvucci et al., 1986). In the dark, Rubisco sites are free of RuBP and readily carbamylate with the available CO 2 . In the light, the production of RuBP can generate a dead-end complex, when it binds to uncarbamylated Rubisco. Thus, in the light, the activation state of Rubisco is determined not by the concentration of CO 2 , except at very low CO 2 , but by the balance between inactivation by RuBP and reactivation by Rca (Salvucci et al., 1985(Salvucci et al., , 1986. To achieve a constant low level of Rubisco activation in both wild-type Arabidopsis and the rwt43 transformant, plants were placed in the dark (i.e. a condition where the activity of Rca does not affect Rubisco activation). After 2 h in the dark at ambient CO 2 , the activation state of Rubisco in both wild-type Arabidopsis and the rwt43 transformant was about 50% (Supplemental Table S1). When plants were transferred from dark to low irradiance, the activation state of Rubisco decreased in wild-type plants but increased in the rwt43 transformant. Thus, the Rca in the rwt43 transformant produced a high Rubisco activation state at low irradiance, both upon transition from high to low irradiance (Table I) and from dark to low irradiance (Supplemental Table S2).
At high irradiance, the amount of the Rubisco substrate, RuBP, was slightly lower in the Arabidopsis rwt43 transformant than in wild-type plants (Supplemental Table S2). At low irradiance, RuBP levels in the rwt43 transformant were less than half the levels in wild-type plants (Supplemental Table S2; Zhang et al., 2002), consistent with the much higher activation state of Rubisco in the transformant (Table I). In contrast, the amounts of Fru-1,6-bisP and triose phosphate in the Table I. Effect of irradiance on the activation state of Rubisco in plants containing different Rca forms Leaf discs were illuminated at 1,000 or 1,800 mmol m 22 s 21 for 30 min (high irradiance) followed by 30 mmol m 22 s 21 for 60 min (low irradiance) and then immediately frozen in liquid nitrogen. Frozen leaf discs were extracted and immediately assayed for initial and total Rubisco activity. The activation state was calculated as the initial activity divided by the total activity at high irradiance multiplied by 100. Values are means 6 SE (n as indicated). Different letters after the values denote significant difference between the values at low and high irradiance (P , 0.05).

Plant
Irradiance n Rubisco Initial Activity Total Activity Activation State rwt43 transformant were similar to the amounts in wild-type plants at both high and low irradiance.

ADP Inhibition of Recombinant b-Rca ATPase Activity
To determine if the difference in ADP sensitivity was an intrinsic characteristic of the enzyme, the effect of various ADP/ATP ratios on the ATPase activity of recombinant b-Rca from Arabidopsis and tobacco was compared ( Fig. 2). At 0.11 ADP/ATP, the activity of the Arabidopsis b-Rca was not significantly different (P . 0.05) from the activity measured in the presence of ATP alone, whereas the activity of the tobacco enzyme was inhibited by about 50%. At 0.33 ADP/ATP, the activity of the Arabidopsis enzyme was inhibited by about 40% compared with 87% for tobacco b-Rca. Thus, the b-Rca from the two species differed markedly in their sensitivity to inhibition by ADP. These differences were consistent with the different effects of ADP on Rubisco activation activity measured in leaf extracts ( Fig. 1) as well as the different response of Rubisco activation to low irradiance measured in planta ( Table I).
Comparison of the amino acid sequences of the b-Rca from Arabidopsis and tobacco showed that the two proteins are 82% identical (Fig. 3). While differences between the two polypeptides are distributed throughout the protein, residues affecting nucleotide binding are likely to fall within the AAA+ motif (Henderson et al., 2011;Stotz et al., 2011). Also, it is likely that ADP sensitivity is conferred by a residue(s) outside the Rubisco recognition domain, since Rca activity in leaf extracts of the Arabidopsis Tob-DAt (S2) transformant was inhibited by physiological ratios of ADP to ATP (Fig. 1).
Differences in the Rca sequence between tobacco and Arabidopsis include two nonconservative residues in the AAA+ module near the nucleotide-binding domain: residue 125 (tobacco numbering) near the Walker A motif and residue 166 near the Walker B motif. These residues were mutated separately in the tobacco b-Rca to the corresponding residue in Arabidopsis. Inhibition of the ATPase activity of the recombinant R125A and N166K proteins by ADP was similar to the wild-type tobacco b-Rca (Fig. 2). Consequently, multiple mutations were introduced in the tobacco b-Rca, changing these two plus 15 other residues to the corresponding residues in Arabidopsis. These changes were targeted to the AAA+ module but outside the Rubisco recognition domain (Fig. 3). No changes were made in the N-terminal domain because previous studies have shown that removal of the first 50 residues of the tobacco Rca does not affect ATP binding or hydrolysis (van de Loo and Salvucci, 1996;Stotz et al., 2011). The mutated enzyme, designated Tob-DAt (n=17) , catalyzed ATP hydrolysis at rates equivalent to wild-type tobacco b-Rca but was much less sensitive to inhibition by ADP, more closely resembling the Arabidopsis than the tobacco b-Rca (Fig. 2).

Light Induction of Photosynthesis in Plants with Different Rca Forms
The rate of photosynthetic induction (i.e. the time required to reach a constant steady-state rate of net CO 2 assimilation upon transition from low to high irradiance) has been attributed to the rate of Rubisco activation by Rca (Woodrow et al., 1996;Mott and Woodrow, 2000). After 1 h at low irradiance, photosynthetic induction upon transition to high irradiance took as long as 30 min in wild-type Arabidopsis, camelina (data not shown), tobacco, and the Arabidopsis Tob-DAt (S2) transformant (Fig. 4). In tobacco, Figure 2. Effect of ADP on the ATPase activity of recombinant Arabidopsis and tobacco b-Rca as well as two point mutants and a 17-residue substitution mutant of tobacco Rca. The rate of ATP hydrolysis was determined in the presence of 5 mM adenine nucleotide at the indicated ratios of ADP to ATP. Rates of ATP hydrolysis are expressed as v i /v c (the inhibited velocity/control velocity, i.e. the rates determined in the presence of ADP relative to the control rate with ATP alone). Control rates were 0.84 6 0.06, 0.52 6 0.01, 0.45 6 0.03, 0.68 6 0.03, and 0.55 6 0.02 units mg 21 protein for Arabidopsis, tobacco, R125A, N166K, and the 17-residue substitution mutant Tob-DAt( n=17 ), respectively. Values are means 6 SE of three to five measurements. Different letters at the base of each bar denote significant differences (P , 0.05) between the rates at each ratio of ADP to ATP.
photosynthetic induction was particularly slow, probably because of the accumulation of 2-carboxyarabinitol-1-P (Table I; Hammond et al., 1998). The rate of photosynthetic induction in these plants increased with decreasing time of exposure to low irradiance.
In contrast to the results with plants containing ADP-sensitive Rca forms, the rate of photosynthetic induction in the Arabidopsis rwt43 transformant was almost instantaneous and was unaffected by the time of exposure at low irradiance. Measurement of chlorophyll fluorescence following an increase from low to moderate (i.e. 200 mmol m 22 s 21 ) irradiance showed that nonphotochemical quenching (NPQ) exhibited a transient increase during photosynthetic induction in wildtype Arabidopsis but not in the rwt43 transformant (Fig. 5).
Relaxation times, determined as the reciprocal of the apparent rate constant during the initial phase of photosynthetic induction (Woodrow and Mott, 1989), were measured in wild-type Arabidopsis and the rwt43 transformant to quantify the rates of photosynthetic induction (Fig. 6). Measurements were conducted at 15°C, 23°C, and 35°C to determine if the regulation of Rca affects photosynthetic induction at suboptimal, optimal, and supraoptimal temperatures (Yamori et al., 2012). Compared with wild-type Arabidopsis, the rate of photosynthetic induction was twice as fast in the rwt43 transformant at 15°C and three times faster at 23°C and 35°C. For both wild-type Arabidopsis and the rwt43 transformant, the rate of photosynthetic induction was fastest at 23°C. This temperature coincides with the temperature optimum of CO 2 assimilation (Kim and Portis, 2005) as well as the optimum for Rca activity in Arabidopsis (Carmo-Silva and . The longer relaxation time at 35°C compared with 23°C in wild-type plants was consistent with inhibition of Rca activity at the higher temperature.

Growth of Plants with Different Rca Forms under Variable Light Environments
Measurements of biomass accumulation indicated that the Arabidopsis rwt43 transformant plants grew more slowly than the wild type in air under control conditions of light and temperature. After 5 weeks, the average dry weight of the wild type and the rwt43 transformant were 95.5 6 4.1 and 67.7 6 3.0 mg, respectively. Even though the rates of photosynthetic induction differed markedly, the light response of photosynthesis was very similar for wildtype Arabidopsis and the rwt43 transformant plants (Supplemental Fig. S2). At saturating irradiance, the CO 2 assimilation rates were higher for the wild-type plants compared with the rwt43 plants, 11.5 and 9.5 mmol m 22 s 21 , respectively. At the irradiance used for growth, the CO 2 assimilation rates were 2.4 6 0.1 and 1.9 6 0.1 mmol m 22 s 21 for the wild type and the rwt43 transformant, respectively. Rca expression in the rwt43 transformant line is about 70% of the level present in wild-type Arabidopsis (Zhang et al., 2002) but certainly not limiting for Rubisco activation (Table I). Residues common to all sequences are in blue. Tobacco residues that are different or missing in the Arabidopsis sequence are in red boldface. Dashed lines immediately above the tobacco sequence indicate the positions of the N terminus and Rubisco recognition domain (dark red). Asterisks indicate the tobacco residues Arg-125 and Asn-166 that were changed individually to the corresponding residues in Arabidopsis. Residues in the tobacco sequence that were changed to the corresponding Arabidopsis residues to make the Tob-DAt (n=17) mutant protein are underlined.
On an area basis, soluble protein was higher in the rwt43 plants than in wild-type plants, 0.28 6 0.01 and 0.25 6 0.02 mg cm 22 , respectively, while chlorophyll content was lower, 0.027 6 0.001 and 0.031 6 0.001 mg cm 22 , respectively. When adjusted for differences in soluble protein, Rubisco total activity was higher on an area basis in the rwt43 plants compared with the wild type (Table I). Kim and Portis (2005) reported that the response of CO 2 assimilation rate to intercellular CO 2 concentration was similar in the rwt43 and wild-type plants, and we confirmed this result in separate experiments (data not shown). The maximal velocity of carboxylation calculated from our measurements of the response of the rate of CO 2 assimilation to the intercellular CO 2 concentration was about 42 mmol m 22 s 21 in both wild-type and rwt43 plants, indicating that Rubisco content was similar in the wild type and the transformant. In addition, Kim and Portis (2005) reported that the temperature response of CO 2 assimilation rate was nearly identical in the rwt43 and wildtype plants.
Arabidopsis wild-type and rwt43 transformant plants were grown under several other conditions to determine if the high activation state of Rubisco at low irradiance and its effect on photosynthetic induction, RuBP levels, and NPQ were detrimental to growth. Compared with plants grown under control conditions (i.e. 10-h photoperiod at 175 mmol m 22 s 21 ), both Arabidopsis wild type and the rwt43 transformant exhibited an increase in shoot dry weight when grown at twice the irradiance (Supplemental Fig. S3). Growth under an alternating irradiance regime (i.e. 420 and 20 mmol m 22 s 21 at 1-h intervals for 15 h) had no adverse effect on the growth of either wild-type Arabidopsis or the rwt43 transformant. In fact, the rwt43 plants responded more positively to growth under a fluctuating light condition than the wild type. In contrast, biomass accumulation decreased for both the wild-type and the rwt43 when darkness was used instead of low irradiance for the alternating light condition. Plants were placed at high irradiance: 1,000 mmol m 22 s 21 for Arabidopsis or 1,800 mmol m 22 s 21 for tobacco. After 20 min, the irradiance was decreased to 30 mmol m 22 s 21 for 5, 15, 30, and 60 min. The irradiance was then increased to 1,000 mmol m 22 s 21 for Arabidopsis or 1,800 mmol m 22 s 21 for tobacco (time zero), and the rate of net CO 2 assimilation was measured continuously thereafter. Measurements were conducted at 23˚C for Arabidopsis or at 28˚C for tobacco. Assimilation rates for tobacco were corrected for differences in stomatal conductance by adjusting the rates to an intercellular CO 2 concentration of 280 mmol mol 21 . No correction was required for Arabidopsis. The relative photosynthetic rate represents the fraction of the maximum rate, measured after 20 min at high irradiance: 9.7 6 0.2, 8.0 6 0.2, 6.6 6 0.1, and 23.4 6 0.9 mmol m 22 s 21 for Arabidopsis wild type, the rwt43 and Tob-DAt (S2) transformants, and tobacco, respectively. Values are means 6 SE of measurements taken with four to five separate plants.

Species Differences in the Regulatory Properties of Rca
The response of Rca activity to physiological ratios of ADP to ATP showed that the regulatory properties of b-Rca in Arabidopsis differed from those of b-Rca in tobacco. In wild-type Arabidopsis, b-Rca is paired with a-Rca, and the interaction of the two isoforms affects the sensitivity of the holoenzyme to inhibition by ADP (Zhang et al., 2001;Wang and Portis, 2006). That the Cys residues in the C-terminal extension are conserved in a-Rca from diverse species, including those produced from separate genes (Supplemental Fig. S1), suggests that redox regulation is necessary for the proper functioning of the a-Rca plus b-Rca holoenzyme, at least under some conditions. In spinach (Spinacia oleracea) and Arabidopsis, the ratio of ADP to ATP is thought to remain constant over a range of irradiances that affect Rubisco activation (Brooks et al., 1988). Thus, in these species, redox sensitivity of a-Rca would be essential for regulating Rubisco activation in response to irradiance.
The b-isoform of Rca does not contain a redoxreactive Cys pair and is unaffected by changes in redox state mediated by thioredoxin f (Zhang and Portis, 1999). Thus, the mechanism for the regulation of Rca in plant species like tobacco that express only the nonredox-regulated b-isoform has been enigmatic. b-Rca from tobacco was acutely sensitive to inhibition by ADP, resembling the response of the Arabidopsis b-Rca when in association with the respective a-Rca. Rca activity was also inhibited by ADP in the Arabidopsis Tob-DAt (S2) transformant, containing a chimeric b-Rca identical to the tobacco enzyme but with the Rubisco recognition domain of Arabidopsis. Mutation Figure 5. Time course of NPQ upon transition from low to moderate irradiance in wild-type Arabidopsis and the rwt43 transformant. Plants were initially dark adapted for 60 min to determine the maximal chlorophyll fluorescence in the dark-adapted leaves (F m ). Plants were then placed at 1,000 mmol m 22 s 21 for 20 min to fully activate Rubisco, and the irradiance was then decreased to 30 mmol m 22 s 21 . After 60 min (time zero), the irradiance was increased to 200 mmol m 22 s 21 and the maximal chlorophyll fluorescence in the light (F m 9) was measured continuously for 15 min at 23˚C. NPQ was calculated as (F m 2 F m 9)/F m 9. Values are means 6 SE of measurements taken with four separate plants. Figure 6. Induction of CO 2 assimilation upon transition from low to high irradiance in Arabidopsis wild-type and rwt43 transformant plants at suboptimal (15˚C), optimal (23˚C), and supraoptimal (35˚C) temperatures. Plants were placed at 1,000 mmol m 22 s 21 . After 20 min, the irradiance was decreased to 30 mmol m 22 s 21 for 60 min. The irradiance was then increased to 1,000 mmol m 22 s 21 (time zero), and the rate of net CO 2 assimilation was measured continuously thereafter. Values are means 6 SE of measurements taken with four to five separate plants.
of 17 residues in the tobacco b-Rca to the corresponding residues in the Arabidopsis b-Rca resulted in a b-  ] that was more similar to Arabidopsis than tobacco b-Rca in its response to physiological ratios of ADP to ATP. Therefore, one or more of these 17 residues (Fig. 3) confer sensitivity to inhibition by ADP to the tobacco b-Rca.
The b-Rca in the Arabidopsis Tob-DAt (S2) transformant is not redox sensitive and strongly resembles the tobacco b-Rca, making it the equivalent of a tobacco b-Rca in an Arabidopsis background. In the Arabidopsis Tob-DAt (S2) plants, Rubisco deactivated under low irradiance, indicating that the ADP/ATP ratio must have decreased to a level that was inhibitory for the chimeric b-Rca. In contrast, Rubisco did not deactivate after transition from high to low irradiance and activated after transition from dark to low irradiance in the Arabidopsis rwt43 transformant, indicating that the ADP/ATP ratio at low irradiance was not inhibitory for the Arabidopsis b-Rca. Taken together, these results indicate that changes in the ADP/ ATP ratio play a much greater role in regulating Rca activity than previously suggested (Brooks et al., 1988;Zhang and Portis, 1999;Ruuska et al., 2000). Moreover, these changes alone might explain the regulation of Rubisco activation in plants like tobacco that contain an ADP-sensitive b-Rca.
It is not clear why different mechanisms have evolved for regulating Rca. Redox regulation of Rca provides fine control over the sensitivity of the holoenzyme to inhibition by ADP (Zhang and Portis, 1999;Zhang et al., 2001). However, this level of regulation does not seem to be essential in all plant species. ATPase activity of cotton and creosote (Larrea tridentata) b-Rca was inhibited by ADP (A.E. Carmo-Silva and M.E. Salvucci, unpublished data), indicating that ADP inhibition of b-Rca varies among plant species and even occurs in species that also express an a-Rca. Similarly, tobacco expresses several different b-Rca proteins (Qian and Rodermel, 1993) that could vary in their sensitivities to inhibition by ADP. However, measurements of the recombinant versions of the products of the two main tobacco Rca genes (i.e. RCA1 and RCA2) showed that their responses to ADP/ATP were similar (A.E. Carmo-Silva and M.E. Salvucci, unpublished data).
Certain plant species like Arabidopsis, camelina, and spinach express equal amounts of a-Rca and b-Rca, while others like rice (Oryza sativa) and wheat (Triticum aestivum) accumulate much more b-Rca than a-Rca Fukayama et al., 2012). In still others like maize, only b-Rca has been detected at the protein level under nonstress conditions Vargas-Suárez et al., 2004), even though the genome contains an open reading frame that encodes for an a-Rca (accession no. NM_001175017.1). Evidence has been presented for the expression of a high-molecular-mass Rca polypeptide, possibly the a-isoform, in some maize cultivars during heat stress (Sánchez de Jiménez et al., 1995;Ristic et al., 2009), similar to the up-regulation of a-Rca expression in rice . Detailed analysis of the kinetic and thermal properties of the various a-Rca and b-Rca forms is necessary to determine how patterns of Rca isoform expression might exert control over enzyme activity.

Light Induction of Photosynthesis and in Vivo Regulation of Rubisco Activation
In previous studies, Mott, Woodrow, and colleagues demonstrated that Rca determines the rate of induction of photosynthetic CO 2 assimilation following an increase in irradiance (Woodrow and Mott, 1989;Woodrow et al., 1996;Mott and Woodrow, 2000). Using transgenic tobacco and Arabidopsis plants with reduced amounts of Rca, they showed that the apparent rate constant for the induction of net CO 2 assimilation was determined by the rate of Rubisco activation by Rca (Mott et al., 1997;Hammond et al., 1998). Similar findings were recently reported for transgenic rice plants with altered amounts of Rca Yamori et al., 2012). The relaxation times (i.e. the reciprocal of the apparent rate constant for the induction of net CO 2 assimilation; Woodrow and Mott, 1989) were shorter in rice plants that overexpressed the b-Rca from barley (Hordeum vulgare) and maize , consistent with the predictions of the model presented by Mott and Woodrow (2000).
In this study, the ADP sensitivity of Rca was correlated with the light modulation of Rubisco activation state and the light induction of photosynthesis. In the Arabidopsis rwt43 transformant, containing only the ADP-insensitive Arabidopsis b-Rca, Rubisco did not deactivate at low irradiance. As a result, the induction of CO 2 assimilation was almost instantaneous, because Rubisco was in a highly activated state even under low irradiance and did not require a period of time to activate when the irradiance was increased.
Rubisco deactivated at low irradiance in tobacco as well as in the Arabidopsis Tob-DAt (S2) transformant, both of which contain a b-Rca that exhibits marked inhibition by ADP. Deactivation of Rubisco at low irradiance was also observed in wild-type Arabidopsis and in camelina, both of which contain a mixed a-Rca and b-Rca holoenzyme that is sensitive to ADP/ATP inhibition in a redox-dependent manner. Because Rubisco deactivated under low irradiance in all of these plants, induction of CO 2 assimilation was much slower upon the transition from low to high irradiance than in the Arabidopsis rwt43 transformant. Also, unlike the rwt43 plants, the rate of induction increased in these plants with increasing time at low irradiance. Thus, by controlling the activation state of Rubisco, the regulatory properties of Rca, and specifically the sensitivity to inhibition by ADP determines the rate of photosynthetic induction upon the transition from low to high irradiance.
Although qualitatively similar, the effect of altered Rca regulation on photosynthetic induction differs mechanistically from the effects obtained by overexpressing Rca Yamori et al., 2012). In transgenic rice that overexpresses barley and maize b-Rca, Rubisco still deactivated at low irradiance, but the higher concentration of Rca promoted faster rates of Rubisco activation after the transition to high irradiance (Yamori et al., 2012). The regulatory properties of rice a-Rca and b-Rca as well as those of maize and barley b-Rca are unknown, but the deactivation of Rubisco at low irradiance even in the Rca-overexpressing lines indicates that Rca in the transgenic plants was inhibited by ADP. In contrast, Rubisco in the Arabidopsis rwt43 transformant maintained a high activation state even under low irradiance because of the altered regulation of Rca. As a result, the rate of photosynthetic induction in the Arabidopsis rwt43 transformant was extremely rapid, because the rate was no longer dependent on the rate of Rubisco activation by Rca.

The Functional Significance of Regulating Rca
The pioneering work of Perchorowicz et al. (1981) showed that when Rubisco deactivates under low irradiance, RuBP levels generally exceed the concentration necessary to saturate Rubisco active sites. By keeping RuBP levels above the binding site concentration, light activation of Rubisco could potentially provide a mechanism to maintain metabolic homeostasis under changing irradiance levels and, thus, avoid lags in the resumption of CO 2 assimilation caused by a slow rate of substrate regeneration through autocatalysis. However, the data from the Arabidopsis rwt43 transformant indicate the opposite. Upon transition from low to high irradiance, no lag in photosynthesis occurred in these plants, even though RuBP levels were much lower than in wild-type plants because of the abnormally high activation state of Rubisco at low irradiance (Zhang et al., 2002). Accordingly, NPQ did not increase in the rwt43 transformant after transition from low to high irradiance, presumably because the rapid increase in CO 2 assimilation provided an outlet for the excess photochemical energy.
Studies with mutants and transgenic plants have shown that Rca is essential for keeping Rubisco active (Salvucci et al., 1985;Mate et al., 1993;Jiang et al., 1994). However, our results comparing growth and photosynthesis in the Arabidopsis wild type and the rwt43 transformant indicate that the only obvious penalty for not regulating Rubisco in response to irradiance was possibly a slight reduction in the rate of CO 2 assimilation at ambient CO 2 (Zhang et al., 2002). However, since Rubisco content was not reduced in the rwt43 transformant, it is unclear why the rate of CO 2 assimilation was reduced in these plants. Perhaps the imbalance between the rates of RuBP consumption and regeneration in the rwt43 transformant, particularly at low irradiance (Supplemental Table S2), exert a feedback effect on the overall turnover rate of the Calvin cycle, making the cycle RuBP limited (Mott et al., 1984).
Biomass accumulation was enhanced under an alternating light regime in the Arabidopsis rwt43 transformant but not in wild-type plants. However, no enhancement occurred when dark was used instead of low irradiance. These results were consistent with the different responses of Rubisco activation to low irradiance versus dark (Supplemental Table S1). Taken together, these data suggest that the faster rate of photosynthetic induction in the transformant translated into a greater carbon gain under fluctuating light conditions. However, developmental factors associated with the alternating light regime or longer photoperiod cannot be totally excluded from consideration. For example, Rca expression is driven by a constitutive promoter in the Arabidopsis rwt43 transformant, whereas Rca expression in the wild type exhibits a strong circadian rhythm (Liu et al., 1996). This difference in the nature of the Rca promoter, as well as differences in the 39 untranslated region between the native Rca gene and the rwt43 transgene, are likely to affect the expression of Rca under the fluctuating light regime (DeRidder et al., 2012) and might influence the expression of other photosynthetic genes. Still, the data presented here suggest a new strategy for enhancing photosynthetic performance under variable light environments based on altering the regulatory properties of Rca to increase the rate of photosynthetic induction.

Plant Material and Growth Conditions
Arabidopsis (Arabidopsis thaliana) wild type (ecotype Columbia) and the transgenic lines rwt43 and Tob-DAt (S2) have been described previously (Zhang et al., 2002;Kumar et al., 2009). Arabidopsis rwt43 is a transformant of the rca mutant that expresses only the Arabidopsis 43-kD b-isoform of Rca (Zhang et al., 2002). Arabidopsis Tob-DAt (S2) , formerly called Arab-Tob, is also a transformant of the rca mutant, but this line expresses a chimeric b-Rca that is similar in sequence to the tobacco (Nicotiana tabacum) Rca except for inclusion of the Rubisco recognition domain from Arabidopsis (Kumar et al., 2009).
Arabidopsis plants were grown in controlled-environment chambers in air at 23°C with a 10-h photoperiod and an irradiance of 175 mmol m 22 s 21 . Plants were initially grown in a CO 2 -enriched atmosphere (2,900 mL L 21 CO 2 ) at 23°C with an 11-h photoperiod and an irradiance of 80 mmol m 22 s 21 . After 2 weeks, plants were transferred to the conditions described above. Camelina (Camelina sativa 'Robinson') plants were grown in air at 23°C/18°C with a 12-h photoperiod and an irradiance of 400 mmol m 22 s 21 . Tobacco 'Petit Havana' SR-1 plants were grown in air at 28°C/23°C with a 16-h photoperiod and an irradiance of 300 mmol m 22 s 21 .
All plants were grown from seed in soil mixture and watered as needed with nutrient solution (Carmo-Silva and Salvucci, 2011) at either full strength (for tobacco) or one-half strength (for all other species). Measurements were conducted on fully expanded leaves of 4-to 5-week-old plants of Arabidopsis and camelina and 5-to 6-week-old plants of tobacco.

Rca Activity in Leaf Extracts
The assay for measuring Rca activity in leaf extracts is described in detail elsewhere . Briefly, leaf discs were excised from intact plants 3 to 4 h after the beginning of the photoperiod, immediately frozen in liquid nitrogen, and stored at 280°C. Freshly frozen leaf material was rapidly extracted at 4°C, desalted by centrifugal gel filtration, and then assayed to determine the rate of ATP-dependent reactivation of the inactive Rubisco-RuBP complex at 25°C. Activation reactions contained 90 mM Tricine-NaOH, pH 8.0, 10 mM NaHCO 3 , and 30 mM MgCl 2 plus or minus 5 mM ATP and an ATP-regenerating system consisting of 5 mM phosphocreatine and 100 units mL 21 phosphocreatine kinase. Where stated, ATP was replaced by a mixture of 5 mM ADP plus ATP at a ratio of 0.11 or 0.33 in the absence of ATPregenerating system. Measurements of ADP concentration at the end of the reaction showed that the ratio of ADP to ATP and the total adenine nucleotide pool did not change markedly during the assay. The rate of Rca activity was calculated as described previously .

Rubisco Activation State in Leaf Extracts
Leaf discs (0.5 cm 2 ) were excised from intact plants in the light, floated on 25 mM MES-NaOH, pH 5.5, at 25°C, and flushed with a humidified gas stream (380 mL L 21 CO 2 and 21% oxygen). For the irradiance treatments, the discs were first illuminated at high irradiance (1,000 or 1,800 mmol m 22 s 21 ) for 20 min followed by low irradiance (30 mmol m 22 s 21 ) for 1 h. For the highirradiance treatment, discs were exposed to high irradiance (1,000 or 1,800 mmol m 22 s 21 ) for an additional 20 min after the low-irradiance treatment. Discs were snap frozen in liquid nitrogen, and samples consisting of two discs each were stored at 280°C. Frozen leaf discs were rapidly extracted and assayed as described previously (Carmo-Silva and Salvucci, 2012). The activation state was calculated as the initial activity divided by the average total activity at high irradiance, multiplied by 100.

Purification and Assay of Rca
Recombinant Arabidopsis, tobacco, and engineered tobacco proteins were purified after expression in Escherichia coli as described previously (van de Loo and Salvucci, 1996). Point mutations were introduced into the tobacco complementary DNA (cDNA) sequence by directed mutagenesis (van de Loo and Salvucci, 1996). For construction of the Tob-DAt (n=17) mutant protein, the tobacco oligonucleotide sequence was engineered to change 17 amino acids in the sequence to the corresponding residues in Arabidopsis Rca. A cDNA with these changes was synthesized commercially (Blue Heron Biotechnology) and expressed in E. coli. The rate of ATP hydrolysis by the purified enzymes was determined by the amount of inorganic phosphate released as described previously (Barta et al., 2011). Inorganic phosphate was determined by the method of Chifflet et al. (1988). The total adenine nucleotide concentration in the assay was 5 mM, presented at a ratio 0.00, 0.11, or 0.33 ADP/ATP. Reaction times were adjusted to ensure that the amount of ATP consumed in the reaction was no more than 10%.

Gas-Exchange and Chlorophyll Fluorescence Measurements
Gas exchange of attached leaves was measured at air levels of CO 2 (380 mL L 21 ) and oxygen, at the temperatures and irradiance indicated in the figure legends, using a portable infrared gas analyzer (LI-6400; LI-COR). For measurement of the induction of CO 2 assimilation, plants were initially placed at high irradiance (1,000 or 1,800 mmol m 22 s 21 ) to fully activate Rubisco. After 20 min, the irradiance level was decreased to 30 (Arabidopsis) or 50 (camelina and tobacco) mmol photons m 22 s 21 (i.e. low irradiance) and maintained at this level for the times indicated in the figure legends. Induction was initiated by instantaneously increasing the irradiance level back to the high level and measuring the rate of CO 2 assimilation over time. Rates of CO 2 assimilation were corrected for lags in stomatal conductance using the response of the rate of CO 2 assimilation to the intercellular CO 2 concentration (Woodrow and Mott, 1989). Relaxation times were calculated as described by Woodrow and Mott (1989). A PAM-2000 chlorophyll fluorometer (Heinz-Walz) was used to measure the induction of NPQ (Carmo-Silva and Salvucci, 2012).

Miscellaneous
Protein concentration in leaf extracts was determined by the method of Bradford (1976). The Rubisco used for determining RuBP levels was purified from Arabidopsis leaves as described previously (Carmo-Silva et al., 2011a).

Statistical Analysis
All values are expressed as means 6 SE of all samples analyzed for each treatment (n as indicated). Statistical comparisons between different treatments were made using either Student's t tests (for two treatments) or ANOVA followed by the Holm-Sidak method for multiple pairwise comparisons (for more than two treatments). P , 0.05 was considered to represent statistical significance.

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Multiple sequence alignment of the C-terminal extension of the Rca a-isoform from several vascular plant species and a moss.
Supplemental Figure S2. Response of CO 2 assimilation rate to irradiance in wild-type Arabidopsis and the rwt43 transformant.
Supplemental Figure S3. Aboveground biomass accumulation in wildtype Arabidopsis and the rwt43 transformant grown under different light regimes.
Supplemental Table S1. Effect of irradiance on the activation state of Rubisco in wild-type Arabidopsis and the rwt43 transformant after initial deactivation in the dark.
Supplemental Table S2. Effect of irradiance on the levels of triose phosphate, Fru-1,6-bisP, and RuBP in wild-type Arabidopsis and the rwt43 transformant.