The catalytic properties of hybrid Rubisco comprising tobacco small and sunflower large subunits mirror the kinetically equivalent source Rubiscos and can support tobacco growth

Abstract

Plastomic replacement of the tobacco (Nicotiana tabacum) Rubisco large subunit gene (rbcL) with that from sunflower (Helianthus annuus; rbcL(S)) produced tobacco(Rst) transformants that produced a hybrid Rubisco consisting of sunflower large and tobacco small subunits (L(s)S(t)). The tobacco(Rst) plants required CO(2) (0.5% v/v) supplementation to grow autotrophically from seed despite the substrate saturated carboxylation rate, K(m), for CO(2) and CO(2)/O(2) selectivity of the L(s)S(t) enzyme mirroring the kinetically equivalent tobacco and sunflower Rubiscos. Consequently, at the onset of exponential growth when the source strength and leaf L(s)S(t) content were sufficient, tobacco(Rst) plants grew to maturity without CO(2) supplementation. When grown under a high pCO(2), the tobacco(Rst) seedlings grew slower than tobacco and exhibited unique growth phenotypes: Juvenile plants formed clusters of 10 to 20 structurally simple oblanceolate leaves, developed multiple apical meristems, and the mature leaves displayed marginal curling and dimpling. Depending on developmental stage, the L(s)S(t) content in tobacco(Rst) leaves was 4- to 7-fold less than tobacco, and gas exchange coupled with chlorophyll fluorescence showed that at 2 mbar pCO(2) and growth illumination CO(2) assimilation in mature tobacco(Rst) leaves remained limited by Rubisco activity and its rate (approximately 11 micromol m(-2) s(-1)) was half that of tobacco controls. (35)S-methionine labeling showed the stability of assembled L(s)S(t) was similar to tobacco Rubisco and measurements of light transient CO(2) assimilation rates showed L(s)S(t) was adequately regulated by tobacco Rubisco activase. We conclude limitations to tobacco(Rst) growth primarily stem from reduced rbcL(S) mRNA levels and the translation and/or assembly of sunflower large with the tobacco small subunits that restricted L(s)S(t) synthesis.

Figure 1.

Growth and phenotype of tobacco^Rst plants compared with wild-type tobacco grown in air containing 0.5% (v/v) CO₂ (see “Material and Methods” for further details). A, Phenotype of wild-type and tobacco^Rst (B and C) plants (tob^Rst1 and tob^Rst4, both germinated from the same seed stock of a T₂ plant derived from previous generations only backcrossed with wild-type pollen) at the juvenile (top sections) and mature growth stage. The number of days since cotyledon emergence is shown. The white arrows indicate the normal oblanceolate leaves (A and B) and irregular oblanceolate leaves (C) analyzed in Figure 7. D, Change in height of the primary axial shoot for wild-type (•, n = 6) and tobacco^Rst plants with one (tob^Rst1, ○, n = 1), two (□, n = 2), or four (tob^Rst4, ▵, n = 1) primary axial shoots. The exponential growth rates (black lines) were modeled to the height data according to the equationwhere A1 is the amplitude, t₁ the time constant (calculated values are shown), and Y₀ is the offset. E, Comparison of a mature wild-type and tob^Rst leaf (F) with the marginal dimpling phenotype in the tob^Rst lines not evident when grown in Suc containing tissue culture medium (G; Svab and Maliga, 1993).

Figure 2.

CO₂ assimilation rates in tobacco^Rst and wild-type tobacco plants and their Rubisco kinetics. A, Gas-exchange measurements of CO₂ assimilation rates in response to intercellular pCO₂ measured at an irradiance of 350 (white symbols) or 950 (black symbols) μmol quanta m⁻² s⁻¹ and leaf temperature of 25°C. Measurements were made on attached leaves from two wild-type plants (wt1, 13.5 cm leaf diameter; wt2, 15 cm leaf diameter containing 33.2 and 30.6 μmol Rubisco sites m⁻², respectively) and two tobacco^Rst plants (tob^Rst1 and tob^Rst2; both 12 cm leaf diameters with 5.8 and 4.9 μmol Rubisco sites m⁻², respectively). The Rubisco limited CO₂ assimilation rates for wt1 (•) and tob^Rst2 (▪) were modeled according to Farquhar et al. (1980) using estimates of maximal ribulose-P₂ dependent carboxylase activity. B, V_c^max, K_m for CO₂ under ambient O₂ levels (K_c^21%O₂), and CO₂/O₂ specificity (S_c/o) measured using leaf protein extracts and purified Rubisco preparations. See “Materials and Methods” for further details. V_c^max measurements in bold italics are those made using leaf tissue from tissue culture grown plants; see Supplemental Figure S3 for details. Superscript a indicates data from Whitney et al. (1999) and superscript b indicates data from Kanevski et al. (1999).

Figure 3.

Rubisco activase abundance and Rubisco content and carbamylation status in comparable young, fully expanded leaves from wild-type tobacco (wt, n = 4) and tobacco^Rst (t^Rst, n = 3) plants of similar physiological age (approximately 35 cm in height) grown in air containing 0.5% (v/v) CO₂. Soluble protein from 2.7 mm² of leaves was separated by SDS-PAGE and stained with Coomassie blue (A) or replicate samples (B) blotted and probed with antibodies raised against spinach Rubisco (Rubisco L antibody), tobacco Rubisco activase (tobacco activase antibody), or tobacco Rubisco S subunit (Rubisco S antibody; Whitney and Andrews, 2001a). C, The content of Rubisco active sites and their carbamylation status (D) measured by [¹⁴C]carboxyarabinitol-P₂ binding/[¹²C]carboxyarabinitol-P₂ exchange under the high-CO₂ growth conditions. See “Materials and Methods” for further details.

Figure 4.

Measurement of the activation constant for tobacco and tobacco^Rst Rubisco. Representative whole leaf gas-exchange measurements of the CO₂ assimilation response for physiologically analogous leaves (12 cm leaf width, 35 cm plant height) from a wild-type tobacco (A) and a tobacco^Rst (B) before and after leaf irradiance was suddenly increased from 110 to 1,200 μmol quanta m⁻² s⁻¹ (time zero). The chamber CO₂ concentration and leaf temperature were set at 250 μbar and 25°C, respectively. Bottom panels show the data replotted as the natural logarithm of the final assimilation rate (A*_f) minus the adjusted assimilation rate (A*) as described (Woodrow and Mott, 1989). See “Materials and Methods” for further details. The rate constant for Rubisco activation (k_a) was calculated from the gradients of the linear second phase fitted to the wild-type (white dashed line) and tobacco^Rst (black dashed line) measurements.

Figure 5.

Rubisco turnover in tobacco and tobacco^Rst leaves. Multiple leaf discs from the same leaf were infiltrated with ³⁵S-Met and chased with 10 mm unlabeled Met for the times shown and the soluble protein from 18 mm² of leaf separated by nondenaturing PAGE. A, Hexadecameric Rubisco from wild-type tobacco (L₈S₈, black arrow) and tobacco^Rst (L^sS^t, white arrow) leaves identified in the gels by Coomassie staining and autoradiography. B, Densitometry measurements of the ³⁵S-labeled Rubiscos during the chase periods (±sd of three separate leaf samples for each time point).

Figure 6.

Plastome organization, transcript content, and translational efficiency of rbcL in comparable light adapted leaves of physiologically analogous tobacco (wt) and tobacco^Rst (t^Rst) plants (approximately 60 cm in height). A, The t^Rst transformants have the tobacco rbcL gene replaced with the corresponding gene from sunflower (rbcL^S) and contain a p-aadA-t gene cassette (p, 16S rDNA rrn promoter and 5′ UTR; t, rps16 3′ UTR; Svab and Maliga, 1993) inserted 174-bp downstream of the rbcL stop codon within the rbcL 3′ UTR (T). The annealing positions of the 5′ΔrbcL probe and the primers LsE and LsH are shown. The dotted lines and numbering (Shinozaki et al., 1986) refer to positioning of the homologously inserted foreign sequence into the tobacco plastome (GenBank accession no. Z00044). P, rbcL promoter and 5′ UTR. B, Blot of total leaf RNA (the amount and relative leaf area used are shown) probed with 5′ΔrbcL, showing the rbcL produced in wt and the mono- (rbcL^S) and bi- (rbcL^S-aadA) cistronic mRNAs produced in the t^Rst plants. Equivalent loading was confirmed by comparing the amount of 16SrRNA transcript (data not shown). C, The relative abundance of the rbcL and rbcL^S mRNAs measured from B and the corresponding Rubisco content measured by [¹⁴C]CABP binding are expressed as a percentage to the average rbcL transcript and Rubisco content in the wt samples. The RTE was obtained by dividing Rubisco content by transcript abundance. For t^Rst, the RTE in parentheses is calculated from the sum of the abundances of the rbcL^S and rbcL^S-aadA transcripts.

Figure 7.

Comparison of Rubisco activase content and Rubisco production in juvenile wild-type tobacco and tobacco^Rst (t^Rst) leaves grown in air containing 0.5% (v/v) CO₂. Total and soluble protein from 0.3 cm² of normal ovate wild-type leaves (n = 6) and the strappy oblanceolate t^Rst leaves (n = 4; refer to white arrows in Fig. 1, A–C) were separated by SDS-PAGE and stained with Coomassie Blue (A) or replicate samples (B) blotted and probed with antibodies raised against spinach Rubisco (Rubisco L antibody) and tobacco Rubisco activase (activase antibody). For comparison, the content of Rubisco active sites measured in the soluble leaf protein by [¹⁴C]CABP binding are shown. C, The abundance of rbcL^S (black) and rbcL^S-aadA (white) transcripts in the t^Rst samples relative to the average amount of rbcL message in wild type measured from RNA blots probed with 5′ΔrbcL (see Fig. 6B for example). The RTE was obtained by dividing Rubisco content by transcript abundance. For tobacco^Rst, the RTE in parentheses is calculated from the sum of the abundances of the rbcL^S and rbcL^S-aadA transcripts.