Hotto, A. | Search bioRxiv

bioRxiv2026

Complementation of a Setaria Rubisco activase mutant with Agave Rubisco activase restores growth and photosynthesis

Photosynthetic carbon assimilation is sensitive to heat stress, with a key component being the thermal sensitivity of Rubisco activase (RCA). One approach to increasing heat tolerance of photosynthesis is to increase the thermotolerance of RCA. Here, we have used a transgenic approach to express RCA{beta} from Agave tequilana (AtRCA) in Setaria viridis. A second line was created where the endogenous SvRCA{beta} was substituted for AtRCA{beta} through complementation of a null mutant, {triangleup}rcaB (AtRCA[bKO]). In vitro assays showed that Agave RCA readily activates Setaria Rubisco, and that its thermostability is higher than that of Setaria RCA. AtRCA[bKO] plants exhibited higher CO2 assimilation at 25{degrees}C compared to WT and AtRCA plants, although Rubisco content and activation did not change. After six hours of heat stress, AtRCA[bKO] plants retained higher CO2 assimilation rates than AtRCA, however, after 24 hours CO2 assimilation declined to similar levels in all lines, suggesting that RCA may not limit carbon assimilation at the conditions tested. Taken together, our results show that substitution of RCA from a CAM plant into a C4 model allows normal growth and supports slightly increased carbon assimilation under control and short-term heat conditions.

plant biology↗

bioRxiv2019

Systematic sequencing of chloroplast transcript termini from Arabidopsis thaliana reveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures

Chloroplast transcription requires numerous quality control steps to generate the complex but selective mixture of accumulating RNAs. To gain insight into how this RNA diversity is achieved and regulated, we systematically mapped transcript ends by developing a protocol called Terminome-Seq. Using Arabidopsis thaliana as a model, we catalogued >215 primary 5 ends corresponding to transcription start sites (TSS), as well as 1,628 processed 5 ends and 1,299 3 ends. While most termini were found in intergenic regions, numerous abundant termini were also found within coding regions and introns, including several major TSS at unexpected locations. A consistent feature was the clustering of both 5 and 3 ends, contrasting with the prevailing description of discrete 5 termini, suggesting an imprecision of the transcription and/or RNA processing machinery. Numerous termini correlated with the extremities of small RNA footprints or predicted stem-loop structures, in agreement with the model of passive RNA protection. Terminome-Seq was also implemented for pnp1-1, a mutant lacking the processing enzyme polynucleotide phosphorylase. Nearly 2,000 termini were altered in pnp1-1, revealing a dominant role in shaping the transcriptome. In summary, Terminome-Seq permits precise delineation of the roles and regulation of the many factors involved in organellar transcriptome quality control.

plant biology↗