Biology subjects

Rajarathinam, Y.

Publications and source records attributed to Rajarathinam, Y..

4 recordsLinked to original sources

bioRxiv2024

Dynamic photosynthetic labelling and carbon-positional mass spectrometry monitor in vivo carbon assimilation rates by ribulose-1,5-bisphosphate carboxylase.

AbstractRibulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) is the most abundant enzyme and CO2 bio-sequestration system on earth. Its in vivo activity is usually determined by 14CO2 incorporation into 3-phosphoglycerate (3PGA). The radiometric analysis of 3PGA does not distinguish carbon positions. Hence, RUBISCO activity that fixes carbon into 1-C position of 3PGA and Calvin-Benson-Bassham (CBB) cycle activities that redistribute carbon into its 2-C and 3-C positions are not resolved. This study aims to provide technology that differentiates between these activities. In source fragmentation of gas chromatography-mass spectrometry (GC- MS) enables paired isotopologue distribution analyses of fragmented substructures and the complete metabolite structure. GC-MS measurements after dynamic photosynthetic 13CO2 labelling allowed quantification of the 13C fractional enrichment (E13C) and molar carbon assimilation rates (A13C) at carbon position 1-C of 3PGA by combination of E13C from carbon positions 2,3-C2 and 1,2,3-C3 with quantification of 3PGA concentrations. We validated the procedure using two GC-time of flight (TOF)-MS instruments, operated at nominal or high mass resolution and tested expected positional labelling of 3PGA by in vivo glycolysis of positional labelled glucose isotopomers. Application to{Delta} gapdh1 and{Delta} gapdh2 mutants of the highly divergent glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from Synechocystis sp. PCC 6803 revealed full inactivation of the CBB cycle with maintained RUBISCO activity in the{Delta} gapdh2 mutant and a CBB cycle modulating role of GAPDH1 under fluctuating CO2 supply. RUBISCO activity in the CBB-deficient{Delta} gapdh2 mutant can re-assimilate CO2 released by catabolic pathways. We suggest that RUBISCO activity in Synechocystis can scavenge carbon loss through the pentose phosphate pathway or other cellular decarboxylation reactions.

plant biology↗

bioRxiv2024

Positional 13C Enrichment Analysis of Aspartate by GC-MS to Determine PEPC Activity In Vivo

Photoautotrophic organisms fix inorganic carbon (Ci) by two enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) and phosphoenolpyruvate carboxylase (PEPC). RUBISCO assimilates Ci (CO2) into the 1-C position of 3-phosphoglycerate (3PGA). The Calvin-Benson-Basham (CBB) cycle redistributes fixed carbon atoms into 2,3-C2 of the same molecule. PEPC uses phosphoenolpyruvate (PEP) derived from 3PGA and assimilates Ci (HCO3-) into 4-C of oxaloacetate (OAA). 1,2,3-C3 of OAA and of its transaminase product aspartate originate directly from 1,2,3-C3 of 3PGA. Positional isotopologue analysis of aspartate, the main downstream metabolite of OAA in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), allows differentiation between PEPC, RUBISCO, and CBB cycle activities within one molecule. We explored in source fragmentation of gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) at nominal mass resolution and GC-atmospheric pressure chemical ionization-MS (GC-APCI-MS) at high mass resolution. This enabled the determination of fractional 13C enrichment (E13C) at each carbon position of aspartate. Two prevailing GC-MS derivatization methods, i.e. trimethylsilylation and tert-butyldimethylsilylation, were evaluated. The method was validated by 13C-isotopomer mixtures of positional labeled aspartic acid. Combination with dynamic 13CO2 labeling of Synechocystis cultures allowed direct measurements of PEPC activity in vivo alongside analyses of RUBISCO and CBB cycle activities. Accurate quantification of aspartate concentration and positional E13C provided molar Ci assimilation rates during the day and night phases of photoautotrophic Synechocystis cultures. The validated method offers several applications to characterize the photosynthetic Ci fixation in different organisms.

biochemistry↗

bioRxiv2022

Remodelled Ribosomes Synthesise a Specific Proteome in Proliferating Plant Tissue during Cold

Plant acclimation to low temperatures occurs through system-wide mechanisms that include proteome shifts, some of which occur at the level of protein synthesis. All proteins are synthesised by ribosomes. Rather than being monolithic, transcript-to-protein translation machines, ribosomes can be selective and cause effective proteome shifts required for successful temperature acclimation. Here, we use apical root meristems of germinating seedlings of the monocotyledonous plant barley as a model to study changes in protein abundance and synthesis rates during cold acclimation. We measure metabolic and physiological parameters that allow us to compare protein synthesis rates in different physiological states, e.g., in cold acclimation compared to the optimal temperature state. We show that ribosomal proteins are independently synthesised and assembled into ribosomal complexes in root proliferative tissue, and assess how the ribo-proteome shifts during cold may be associated with changes in synthesis and accumulation of macromolecular complexes. We demonstrate that translation initiation is the limiting step during cold acclimation and based on our data propose a model of a ribosomal code that depends on a reconfigured ribosome population, where as a mode of cold acclimation, specific ribosomal protein compositions may confer selective association capabilities between 60S subunits and 48S initiation complexes.

plant biology↗

bioRxiv2021

Homologs of circadian clock proteins impact the metabolic switch between light and dark growth in the cyanobacterium Synechocystis sp. PCC 6803

The putative circadian clock system of the facultative heterotrophic cyanobacterial strain Synechocystis sp. PCC 6803 comprises the following three Kai-based systems: a KaiABC-based potential oscillator that is linked to the SasA-RpaA two-component output pathway and two additional KaiBC systems without a cognate KaiA component. Mutants lacking the genes encoding the KaiAB1C1 components or the response regulator RpaA show reduced growth in light/dark cycles and do not show heterotrophic growth in the dark. In the present study, the effect of these mutations on central metabolism was analyzed by targeted and nontargeted metabolite profiling. The strongest metabolic changes were observed in the dark in {Delta}rpaA and, to a lesser extent, in the {Delta}kaiAB1C1 mutant. These observations included the overaccumulation of 2-phosphoglycolate, which correlated with the overaccumulation of the RbcL subunit in the mutants, and taken together, these data suggest enhanced RubisCO activity in the dark. The imbalanced carbon metabolism in the {Delta}rpaA mutant extended to the pyruvate family of amino acids, which showed increased accumulation in the dark. Hence, the deletion of the response regulator rpaA had a more pronounced effect on metabolism than the deletion of the kai genes. The larger impact of the rpaA mutation is in agreement with previous transcriptomic analyses and likely relates to a KaiAB1C1-independent function as a transcription factor. Collectively, our data demonstrate an important role of homologs of clock proteins in Synechocystis for balanced carbon and nitrogen metabolism during light-to-dark transitions.

microbiology↗