PROTON GRADIENT REGULATION 5 (PGR5) is essential for generating proton motive force across thylakoid membranes in C3 plants and supporting photoprotection under fluctuating light conditions. It is proposed that this function is achieved by regulating cyclic electron flow around Photosystem I. During the evolutionary transition from C3 to C4 photosynthesis, the leaf abundance of PGR5 has increased, coinciding with a rise in the cyclic electron flow rate. To investigate the contribution of PGR5 to photoprotection in C4 photosynthesis, we generated model C4 monocot Setaria viridis with null pgr5 alleles. We show that plants lacking PGR5 struggle to establish proton motive force and energy-dependent non-photochemical quenching (qE) at higher irradiances during instantaneous measurements. This leads to a progressive decline in maximum Photosystem I activity when leaves are exposed to repeated cycles of high irradiance. Additionally, plants without PGR5 exhibit severely reduced growth and photosynthesis compared to wild type plants when grown under fluctuating daylight but not under constant daylight. In the absence of PGR5, a slower-relaxing, zeaxanthin-dependent form of non-photochemical quenching supports growth under fluctuating light, albeit at the cost of reduced photochemical efficiency and assimilation rate. Our findings underscore the role of PGR5 in enabling efficient C4 photosynthesis under fluctuating light by establishing proton motive force for the rapid upregulation of qE and preventing photodamage to the electron transport machinery. This study highlights the importance of various non-photochemical quenching mechanisms for C4 photosynthesis and emphasises the role of PGR5 in the evolution of C4 plants. ### Competing Interest Statement The authors have declared no competing interest.

The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO2-concentrating mechanism. BCT1 could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis.

Plant oil production has been increasing continuously in the past decade. There has been significant investment in the production of high biomass plants with elevated oil content. We recently showed that the expression of Arabidopsis thaliana WRI1 and DGAT1 genes increase oil content by up to 15% in leaf dry weight tissue. However, triacylglycerols in leaf tissue are subject to degradation during senescence. In order to better package the oil, we expressed a series of lipid droplet proteins isolated from bacterial and plant sources in Nicotiana benthamiana leaf tissue. We observed further increases in leaf oil content of up to 2.3-fold when we co-expressed Sesamum indicum Oleosin L with AtWRI1 and AtDGAT1. Biochemical assays and lipid droplet visualization with confocal microscopy confirmed the increase in oil content and revealed a significant change in the size and abundance of lipid droplets.

Protocol to purify 6xHis-tagged recombinant proteins expressed in E. coli using Ni-NTA pull-down.

We describe our efforts at developing a one-step quantitative reverse-transcription (qRT)-PCR protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA directly from saliva samples, without RNA purification. We find that both heat and the presence of saliva impairs the ability to detect synthetic SARS-CoV-2 RNA. Buffer composition (for saliva dilution) was also crucial to effective PCR detection. Using the SG2 primer pair, designed by Sigma-Aldrich, we were able to detect the equivalent of 1.7×10 6 viral copies per mL of saliva after heat inactivation; approximately equivalent to the median viral load in symptomatic patients. This would make our assay potentially useful for rapid detection of high-shedding infected individuals. We also provide a comparison of the PCR efficiency and specificity, which varied considerably, across 9 reported primer pairs for SARS-CoV-2 detection. Primer pairs SG2 and CCDC-N showed highest specificity and PCR efficiency. Finally, we provide an alternate primer pair to use as a positive control for human RNA detection in SARS-CoV-2 assays, as we found that the widely used US CDC primers (targeting human RPP30 ) do not span an exon-exon junction and therefore does not provide an adequate control for the reverse transcription reaction.