Targeted quantitative analysis of a diurnal RuBisCO subunit expression and translation profile in Chlamydomonas reinhardtii introducing a novel Mass Western approach

Highlights

• RuBisCo subunit stoichiometry during a diurnal cycle on Chlamydomonas reinhardtii

• Integrative and quantitative approach of transcriptomic and proteomic analyses

• Novel Mass Western approach

• Proteins and transcripts show diurnal time course.

• RuBisCO-subunits LSU and SSU are differentially expressed

Abstract

RuBisCO catalyzes the rate-limiting step of CO₂ fixation in photosynthesis. Hypothetical mechanisms for the regulation of rbcL and rbcS gene expression assume that both large (LSU) and small (SSU) RuBisCO subunit proteins (RSUs) are present in equimolar amounts to fit the 1:1 subunit stoichiometry of the holoenzyme. However, the actual quantities of the RSUs have never been determined in any photosynthetic organism. In this study the absolute amount of rbc transcripts and RSUs was quantified in Chlamydomonas reinhardtii grown during a diurnal light/dark cycle. A novel approach utilizing more reliable protein stoichiometry quantification is introduced. The rbcL:rbcS transcript and protein ratios were both 5:1 on average during the diurnal time course, indicating that SSU is the limiting factor for the assembly of the holoenzyme. The oscillation of the RSUs was 9 h out of phase relative to the transcripts. The amount of rbc transcripts was at its maximum in the dark while that of RSUs was at its maximum in the light phase suggesting that translation of the rbc transcripts is activated by light as previously hypothesized. A possible post-translational regulation that might be involved in the accumulation of a 37-kDa N-terminal LSU fragment during the light phase is discussed.

Biological significance

A novel MS based approach enabling the exact stoichiometric analysis and absolute quantification of protein complexes is presented in this article. The application of this method revealed new insights in RuBisCO subunit dynamics.

Introduction

During the last decade, quantitative analysis of proteins and their relative changes in abundance in response to environmental perturbation has become routine. Absolute quantification, however, remains challenging. The most accurate method to date is the utilization of the stable isotope dilution technique [1] using isotopic labeled standard peptides of known amounts in combination with the SRM approach. This method has initially been used by Barnidge and colleagues [2] for the targeted absolute protein quantification of G protein-coupled receptor rhodopsin using SDS-PAGE and was called Mass Western [3], [4] for the targeted absolute protein quantification of complex in-solution digests. Different protein isoforms can be distinctively quantified to yield absolute amounts from a complex sample with a single MS analysis [3], [5]. However, equimolarity and accurate quantity of the standard peptides remain critical factors for determination of precise protein complex stoichiometry. Holzmann and colleagues [6] described the equimolarity through equalizer peptide (EtEP) strategy based on mTRAQ. For this method, each standard peptide is coupled to an equalizer peptide (EP). Labeling is introduced to peptides after synthesis using mTRAQ. Alternatively, labeled synthesized peptides with amino acid quantitative analysis are available. Here, we introduce a novel Mass Western strategy that uses cross-concatenated synthetic peptides from different RSU protein subunits including an EP and introducing a quantifier peptide (QP).

The Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the key CO₂-fixing enzyme in photosynthetic eukaryotes. RuBisCO has been described as one of the most inefficient enzymes due to a very low catalytic turnover rate and competing carboxylase and oxygenase activities. RuBisCO therefore limits photosynthetic CO₂ fixation and it is generally accepted that chloroplasts must accumulate large quantities of RuBisCO to sustain sufficient rates of carbohydrate synthesis [7]. Besides its role in carbon assimilation this enzyme complex became of main focus for its plant nitrogen (N) use efficiency [8]. RuBisCO degradation into amino acids leads to N re-utilization for the de novo protein synthesis.

RuBisCO consists of eight large and eight small protein subunits in both higher and lower plants. The large subunit (LSU) is encoded by the rbcL gene localized in the chloroplastic genome while several variants of the small subunit (SSU) are encoded in the nuclear genome by the rbcS gene family. The LSU and SSU subunits are assumed to be present in equimolar quantities within plant cells to match the one-to-one RuBisCO subunit stoichiometry [9]. The localization of RuBisCO genes in two different cellular compartments and the assumed one-to-one stoichiometric balance of its subunits suggest the existence of mechanisms to tightly coordinate the expression of rbcL and rbcS genes [10]. Although the coordinated synthesis of the subunits has been observed [11], the absolute concentrations of LSU and SSU have never been determined. The absolute concentration of LSU and SSU at a certain time point integrates several parameters such as the stability as well as the rates of synthesis and decay of each polypeptide and is therefore crucial to a quantitative understanding of RuBisCO gene expression.

Diurnal cycling of LSU and SSU cellular concentration with peak abundance levels during the photoperiod has been recently suggested by an analysis of protein relative expression change in rice leaves [12]. However, a transcript study [13] reported that in Arabidopsis, grown in a light/dark photoperiod, rbcS mRNA exhibits a diurnal pattern of expression, with peak abundance occurring soon after onset of light and minimum levels at the end of the light period. Thus, diurnal oscillation of relative rbcS mRNA levels in Arabidopsis occurred in an inverse time frame to the SSU oscillation in rice leaves. Consistently with this observation, several studies of rbcL gene expression in Chlamydomonas reinhardtii [14], [15], [16] indicated that rbcL mRNA levels are not directly correlated to the amount of LSU. However, the diurnal variations of the absolute amount of rbcL and rbcS transcript and protein have never been characterized altogether in any plant model.

The aim of this study was to determine the absolute amounts of rbcL and rbcS transcripts and proteins in C. reinhardtii grown during a diurnal time course. The green algae C. reinhardtii is an interesting model system for the study of the photosynthetic apparatus, because mixotrophic cells under low light have been shown to achieve growth rates similar to photoautotrophic counterparts when supplied with acetate [17]. The particular strain CC503 was used to investigate phototaxis and biofuel production and its genome sequence [18] was used to reconstruct a genome-scale metabolic network [19].

We present a novel conceptual approach of the Mass Western [5] that allowed us to determine absolute protein concentrations with accurate stoichiometry as well as diurnal protein translation changes of the different RuBisCO subunit proteins (RSUs).