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. 2012 Mar;63(5):2159-70.
doi: 10.1093/jxb/err434. Epub 2012 Jan 5.

RBCS1A and RBCS3B, two major members within the Arabidopsis RBCS multigene family, function to yield sufficient Rubisco content for leaf photosynthetic capacity

Masanori Izumi  1 , Honami Tsunoda, Yuji Suzuki, Amane Makino, Hiroyuki Ishida
Affiliations

RBCS1A and RBCS3B, two major members within the Arabidopsis RBCS multigene family, function to yield sufficient Rubisco content for leaf photosynthetic capacity

Masanori Izumi et al. J Exp Bot. 2012 Mar.

Abstract

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (RBCS) is encoded by a nuclear RBCS multigene family in many plant species. The contribution of the RBCS multigenes to accumulation of Rubisco holoenzyme and photosynthetic characteristics remains unclear. T-DNA insertion mutants of RBCS1A (rbcs1a-1) and RBCS3B (rbcs3b-1) were isolated among the four Arabidopsis RBCS genes, and a double mutant (rbcs1a3b-1) was generated. RBCS1A mRNA was not detected in rbcs1a-1 and rbcs1a3b-1, while the RBCS3B mRNA level was suppressed to ∼20% of the wild-type level in rbcs3b-1 and rbcs1a3b-1 leaves. As a result, total RBCS mRNA levels declined to 52, 79, and 23% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. Rubisco contents showed declines similar to total RBCS mRNA levels, and the ratio of Rubisco-nitrogen to total nitrogen was 62, 78, and 40% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. The effects of RBCS1A and RBCS3B mutations in rbcs1a3b-1 were clearly additive. The rates of CO(2) assimilation at ambient CO(2) of 40 Pa were reduced with decreased Rubisco contents in the respective mutant leaves. Although the RBCS composition in the Rubisco holoenzyme changed, the CO(2) assimilation rates per unit of Rubisco content were the same irrespective of the genotype. These results clearly indicate that RBCS1A and RBCS3B contribute to accumulation of Rubisco in Arabidopsis leaves and that these genes work additively to yield sufficient Rubisco for photosynthetic capacity. It is also suggested that the RBCS composition in the Rubisco holoenzyme does not affect photosynthesis under the present ambient [CO(2)] conditions.

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Figures

Fig. 1.
Fig. 1.
Identification of rbcs1a-1, rbcs3b-1, and rbcs1a3b-1 mutants. (A) Genomic structures of RBCS1A and RBCS3B loci and T-DNA insertion sites. White and black boxes represent exons. The white boxes represent the untranslated regions and the black boxes represent the coding regions in exons. Grey boxes represent T-DNA. Arrows represent the positions of primer pairs used for RT-PCR analysis. (B) RT-PCR analysis for investigating RBCS1A and RBCS3B mRNA accumulation in leaves of rbcs mutants. Total RNA was isolated from leaves of wild-type, rbcs1a-1, rbcs3b-1, and rbcs1a3b-1 plants and subjected to RT-PCR analysis using gene-specific primers. PCR products were separated by electrophoresis, stained with SYBR Green I, and detected by a fluorescence image analyser. 18S rRNA was used as an internal control.
Fig. 2.
Fig. 2.
Phenotypes of rbcs mutant plants under long-day growth conditions. (A) Photograph of wild-type, rbcs1a-1, rbcs3b-1, and rbcs1a3b-1 plants at 23 d after sowing. (B, C) Shoot fresh weight (B) and the maximum efficiency of PSII (Fv/Fm) (C) of wild-type and each rbcs mutant when wild-type, rbcs1a-1, and rbcs3b-1 plants initiated bolting (23 d after sowing) or rbcs1a3b-1 plants initiated bolting (33 d after sowing). Data represent the means ±SE (n=4). Statistical analysis was performed by Tukey’s test; columns with the same letter were not significantly different (P ≤ 0.05).
Fig. 3.
Fig. 3.
Transcript abundances of each member within the RBCS multigene family in leaves of rbcs mutants. Total RNA was isolated from leaves of wild-type, rbcs1a-1, rbcs3b-1, and rbcs1a3b-1 plants when each plant initiated bolting, and subjected to quantitative real-time RT-PCR analysis using gene-specific primers for determination of transcript abundances of RBCS1A, RBCS1B, RBCS2B, and RBCS3B. Data represent the means ±SE (n=4). Statistical analysis was performed by Tukey’s test; columns with the same letter were not significantly different (P ≤ 0.05).
Fig. 4.
Fig. 4.
Effects of RBCS mutations on the transcript abundances of total RBCS and RbcL. Total RNA was isolated from leaves of wild-type, rbcs1a-1, rbcs3b-1, and rbcs1a3b-1 plants when each plant initiated bolting, and subjected to quantitative real-time RT-PCR analysis using gene-specific primers for determination of transcript abundances of RBCS (A) and RbcL (B). The amount of total RBCS mRNA is a combination of the RBCS members shown in Fig. 3. All mRNA levels are shown as relative levels of the wild-type level. 18S rRNA was used as an internal control (C). Data represent the means ±SE (n=4). Statistical analysis was performed by Tukey’s test; columns with the same letter were not significantly different (P ≤ 0.05).
Fig. 5.
Fig. 5.
Effects of RBCS mutations on leaf Rubisco content. The amounts of chlorophyll, soluble protein, nitrogen, and Rubisco were determined in leaves of wild-type, rbcs1a-1, rbcs3b-1, and rbcs1a3b-1 plants when each plant initiated bolting. The ratios of chlorophyll-nitrogen (Chl-N/total N), soluble protein-nitrogen (Soluble protein-N/total N), and Rubisco-nitrogen (Rubisco-N/total N) to total nitrogen were calculated. Data represent the means ±SE (n=4). Statistical analysis was performed by Tukey’s test; columns with the same letter were not significantly different (P ≤ 0.05).
Fig. 6.
Fig. 6.
SDS–PAGE analysis of Rubisco in leaves of rbcs mutants. Total soluble protein (10 μg for gel stain, 1 μg for immunoblotting) extracted from mature leaves of wild-type, rbcs1a-1, rbcs3b-1, and rbcs1a3b-1 plants was separated by SDS–PAGE, and either stained with Coomassie Brilliant Blue R-250 (gel stain) or detected by immunoblotting with anti-RBCS antibodies (anti-RBCS). White arrowheads indicate the RBCSB protein derived from RBCS1B, RBCS2B, and RBCS3B genes, and black arrowheads indicate the RBCSA protein derived from the RBCS1A gene. The sizes of molecular mass markers (M. M.) are indicated at the left of the stained gel.
Fig. 7.
Fig. 7.
Effects of RBCS mutations on the rate of CO2 assimilation versus intercellular CO2 partial pressure under light saturation. (A) The rate of CO2 assimilation (A) and intercellular CO2 partial pressure (pCi) were measured under light saturation (800 μmol quanta m−2 s−1) in mature leaves of the wild type (circles), rbcs1a-1 (squares), rbcs3b-1 (triangles), and rbcs1a3b-1 (rhombuses) at 5 d after bolting. The arrowhead indicates the point obtained at the present ambient CO2 partial pressure (pCa=40 Pa). (B) The rates of CO2 assimilation are shown as relative levels of the rate at pCi=20 Pa. Data represent the means ±SE (n=6–8).
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