The regulatory properties of Rubisco activase differ among species and affect photosynthetic induction during light transitions

Abstract

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 fine tuned by redox regulation of the α-isoform. To elucidate the mechanism for Rca regulation in species containing only the redox-insensitive β-isoform, the response of activity to ADP was characterized for different Rca forms. When assayed in leaf extracts, Rubisco activation was significantly inhibited by physiological ratios of ADP to ATP in species containing both α-Rca and β-Rca (Arabidopsis and camelina [Camelina sativa]) or just the β-Rca (tobacco [Nicotiana tabacum]). However, Rca activity was insensitive to ADP inhibition in an Arabidopsis transformant, rwt43, which expresses only Arabidopsis β-Rca, although not in a transformant of Arabidopsis that expresses a tobacco-like β-Rca. ATP hydrolysis by recombinant Arabidopsis β-Rca was much less sensitive to inhibition by ADP than recombinant tobacco β-Rca. Mutation of 17 amino acids in the tobacco β-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 CO2 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 fluctuating 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 variable light environments.

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 β-Rca and the line Tob-ΔAt_(S2) expresses a chimeric β-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 ± 0.02, 0.20 ± 0.00, 0.04 ± 0.01, 0.10 ± 0.01, and 0.13 ± 0.01 Rubisco sites activated per min for Arabidopsis wild type, rwt43, Tob-ΔAt_(S2), tobacco, and camelina, respectively. Values are means ± 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.

Figure 2.

Effect of ADP on the ATPase activity of recombinant Arabidopsis and tobacco β-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 ± 0.06, 0.52 ± 0.01, 0.45 ± 0.03, 0.68 ± 0.03, and 0.55 ± 0.02 units mg⁻¹ protein for Arabidopsis, tobacco, R125A, N166K, and the 17-residue substitution mutant Tob-ΔAt(_n=17), respectively. Values are means ± 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.

Figure 3.

Multiple sequence alignment of β-Rca from tobacco, Arabidopsis, and two tobacco-Arabidopsis chimeras, Tob-ΔAt_(S2) and Tob-ΔAt_(n=17). 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-ΔAt_(n=17) mutant protein are underlined.

Figure 4.

Induction of CO₂ assimilation upon the transition from low to high irradiance in plants with different Rca forms. Plants were placed at high irradiance: 1,000 μmol m⁻² s⁻¹ for Arabidopsis or 1,800 μmol m⁻² s⁻¹ for tobacco. After 20 min, the irradiance was decreased to 30 μmol m⁻² s⁻¹ for 5, 15, 30, and 60 min. The irradiance was then increased to 1,000 μmol m⁻² s⁻¹ for Arabidopsis or 1,800 μmol m⁻² s⁻¹ for tobacco (time zero), and the rate of net CO₂ 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 CO₂ concentration of 280 μmol mol⁻¹. 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 ± 0.2, 8.0 ± 0.2, 6.6 ± 0.1, and 23.4 ± 0.9 μmol m⁻² s⁻¹ for Arabidopsis wild type, the rwt43 and Tob-ΔAt_(S2) transformants, and tobacco, respectively. Values are means ± se of measurements taken with four to five separate plants.

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 μmol m⁻² s⁻¹ for 20 min to fully activate Rubisco, and the irradiance was then decreased to 30 μmol m⁻² s⁻¹. After 60 min (time zero), the irradiance was increased to 200 μmol m⁻² s⁻¹ and the maximal chlorophyll fluorescence in the light (F_m′) was measured continuously for 15 min at 23°C. NPQ was calculated as (F_m − F_m′)/F_m′. Values are means ± se of measurements taken with four separate plants.

Figure 6.

Induction of CO₂ 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 μmol m⁻² s⁻¹. After 20 min, the irradiance was decreased to 30 μmol m⁻² s⁻¹ for 60 min. The irradiance was then increased to 1,000 μmol m⁻² s⁻¹ (time zero), and the rate of net CO₂ assimilation was measured continuously thereafter. Values are means ± se of measurements taken with four to five separate plants.