The authors introduced Rubisco activase (RCA) from the CAM species Agave tequilana (referred to as AtRCA) into the C4 model plant Setaria viridis. Two classes of transgenic material were produced: lines that express AtRCA in the presence of the native Setaria RCA, and a complementation line in which the endogenous SvRCAβ was replaced by AtRCA through rescue of a null mutant (designated ΔrcaB; the complemented line is referred to here as AtRCA[bKO]). The study aimed to determine whether AtRCA can functionally activate Setaria Rubisco and whether the greater thermostability reported for AtRCA delivers improved photosynthetic performance or heat tolerance in Setaria.
A null mutant of the Setaria RCAβ gene (ΔrcaB) was complemented with AtRCA to produce the AtRCA[bKO] line. Separate transgenic lines expressing AtRCA in a wild-type background were also generated. The source material reports that AtRCA[bKO] plants showed restored growth, indicating that Agave RCA can substitute for the endogenous RCAβ to support normal development in Setaria viridis. Specific molecular constructs, transformation methods, and selection regimes were not detailed in the source abstract.
Biochemical in vitro assays demonstrated that Agave RCA readily activates Setaria Rubisco, confirming cross-species compatibility of RCA activity toward Rubisco from Setaria. Comparative thermostability assays indicated that AtRCA has higher thermostability than the native Setaria RCA. These in vitro findings provided the mechanistic basis for testing whether AtRCA could improve photosynthetic performance or heat tolerance in planta.
Under control growth conditions at 25°C, the AtRCA[bKO] complementation line exhibited higher CO2 assimilation rates than both wild-type and the AtRCA transgenic lines that retained native RCA. This increase in CO2 assimilation occurred in the absence of detectable changes in total Rubisco content or in the measured Rubisco activation state, according to the summary presented. The observed higher assimilation in AtRCA[bKO] therefore did not appear to result from increased Rubisco abundance or a higher steady-state activation level, based on the reported measurements.
Plants were exposed to heat stress and CO2 assimilation was tracked. After six hours of heat stress, AtRCA[bKO] plants retained higher CO2 assimilation rates compared with the AtRCA transgenic line, consistent with the higher thermostability measured for AtRCA in vitro. However, after 24 hours of heat stress CO2 assimilation declined to similar levels in all lines tested (AtRCA[bKO], AtRCA transgenics, and wild-type). The authors interpret these dynamics to mean that AtRCA can confer an advantage under short-term heat exposure but that this advantage does not persist after prolonged stress under the experimental conditions used.
The combined results show that substitution of RCA from a CAM plant into a C4 model can support normal growth and yield modest increases in carbon assimilation under control and short-term heat conditions. Higher thermostability of AtRCA and its capacity to activate Setaria Rubisco are consistent with the observed short-term maintenance of CO2 assimilation during early heat exposure. However, because CO2 assimilation converged across lines after 24 hours of heat, the data presented suggest that RCA thermostability alone may not be the limiting factor for sustained carbon assimilation under the tested heat regime. Other components of the photosynthetic apparatus or downstream stress responses may constrain long-term maintenance of photosynthesis during extended heat.
This report is a preprint and has not been certified by peer review. The abstract summarizes the main findings but does not report experimental details such as transformation methods, replicate numbers, statistical analyses, exact temperature regimes used for heat stress, growth conditions, or full biochemical datasets. Those details were not reported in the source abstract and would need to be consulted in the full preprint for comprehensive assessment. The authors declared no competing interests in the source.