In situ community transcriptomics illuminates CO₂-fixation potentials and supporting roles of phagotrophy and proton pump in plankton in a subtropical marginal sea

Lineage-wise physiological activities of plankton communities in the ocean are important but challenging to characterize. Here, we conducted whole-assemblage metatranscriptomic profiling at continental shelf and slope sites in the South China Sea to investigate carbon fixation potential in different lineages. RuBisCO expression, the proxy of Calvin carbon fixation (CCF) potential, was mainly contributed by Bacillariophyta, Chlorophyta, Cyanobacteria, and Haptophyta, which was differentially affected by environmental factors among lineages. CCF potential exhibited positive or negative correlations with phagotrophy gene expression, suggesting phagotrophy possibly enhances or complements CCF. Our data also reveal significant non-Calvin carbon fixation (NCF) potential, as indicated by the active expression of genes in all five currently recognized NCF pathways, mainly contributed by Flavobacteriales, Alteromonadales, and Oceanospirillales. Furthermore, in Flavobacteriales, Alteromonadales, Pelagibacterales, and Rhodobacterales, NCF potential was positively correlated with proton-pump rhodopsin (PPR) expression, suggesting that NCF might be energetically supported by PPR. The novel insights into the lineage-differential potential of carbon fixation, widespread mixotrophy, and PPR as an energy source for NCF lay a methodological and informational foundation for further research to understand carbon fixation and the trophic landscape in the ocean. IMPORTANCE Marine plankton plays an important role in global carbon cycling and climate regulation. Phytoplankton and cyanobacteria fix CO2 to produce organic compounds using solar energy and mainly by the Calvin cycle, whereas autotrophic bacteria and archaea may fix CO2 by non-Calvin cycle carbon fixation pathways. How active individual lineages are in carbon fixation and mixotrophy, and what energy source bacteria may employ in non-Calvin carbon fixation, in a natural plankton assemblage are poorly understood and underexplored. Using metatranscriptomics, we studied carbon fixation in marine plankton with lineage resolution in tropical marginal shelf and slope areas. Based on the sequencing results, we characterized the carbon fixation potential of different lineages and assessed Calvin- and non-Calvin- carbon fixation activities and energy sources. Data revealed a high number of unigenes (4.4 million), lineage-dependent differential potentials of Calvin carbon fixation and responses to environmental conditions, major contributors of non-Calvin carbon fixation, and their potential energy source.