Biology subjects

Stone, W. D.

Publications and source records attributed to Stone, W. D..

4 recordsLinked to original sources

bioRxiv2025

DISCOVERY AND BIOCHEMICAL CHARACTERIZATION OF A FUNGAL ICE NUCLEATION PROTEIN FROM PODILA CLONOCYSTIS

Biological ice nucleation plays a pivotal role in atmospheric processes, yet the molecular basis of fungal ice nucleation remains poorly understood compared to bacterial systems. Here, we report the biochemical characterization of an ice nucleation protein (PcINP) from a soil-dwelling fungus Podila clonocystis, not previously reported to produce ice nuclei. Using sequence similarity network analysis, we identified PcINP as a putative fungal homolog of bacterial ice nucleation proteins and confirmed its function through recombinant expression in Escherichia coli. We probe the function of PcINP structure through domain truncations and demonstrate that a poorly structured N-terminal region is not necessary for ice nucleation activity and can be functionally replaced with an expression enhancing SUMO fusion tag. Finally, we observe both monomeric and aggregated PcINP in E. coli lysates using SEC-MALS but are unable to distinguish their ice nucleation activity pointing to an unknown in vitro aggregation mechanism. Our findings establish PcINP within the emerging class fungal ice nucleation protein with distinct structural features and high stability, expanding the known diversity of biological ice nucleators and highlighting their potential for environmental and biotechnological applications.

biochemistry↗

bioRxiv2025

Modification of Non-photochemical Quenching Pathways in the C4 Model Plant Setaria viridis Revealed Shared and Unique Photoprotection Mechanisms as Compared to C3 Plants

Light is essential for photosynthesis; however, excess light can increase the accumulation of photoinhibitory reactive oxygen species that reduce photosynthetic efficiency. Plants have evolved photoprotective non-photochemical quenching (NPQ) pathways to dissipate excess light energy. In tobacco and soybean (C3 plants), overexpression of three NPQ genes, violaxanthin de-epoxidase (VDE), Photosystem II Subunit S (PsbS), and zeaxanthin epoxidase (ZEP), hereafter VPZ, resulted in faster NPQ induction and relaxation kinetics, and increased crop yields in field conditions. NPQ is well-studied in C3 plants; however, NPQ and the translatability of the VPZ approach in C4 plants is poorly understood. The green foxtail Setaria viridis is an excellent model to study photosynthesis and photoprotection in C4 plants. To understand the regulation of NPQ and photosynthesis in C4 plants, we performed transient overexpression in Setaria protoplasts and generated (and employed) stable transgenic Setaria plants overexpressing one of the three Arabidopsis NPQ genes or all three NPQ genes (AtVPZ lines). Overexpressing (OE) AtVDE and AtZEP in Setaria produced similar results as in C3 plants, with increased or reduced zeaxanthin (thus NPQ), respectively. However, overexpressing AtPsbS appeared to be challenging in Setaria, with largely reduced NPQ in protoplasts and under-represented homozygous AtPsbS-OE lines, potentially due to competitive and tight heterodimerization of AtPsbS and SvPsbS proteins. Furthermore, Setaria AtVPZ lines had increased zeaxanthin, faster NPQ induction, higher NPQ level, but slower NPQ relaxation. Despite this, AtVPZ lines had improved growth as compared to wildtype under several conditions, especially high temperatures, which is not related to the faster relaxation of NPQ but may be attributable to increased zeaxanthin and NPQ in C4 plants. Our results identified shared and unique characteristics of the NPQ pathway in C4 model Setaria as compared to C3 plants and provide insights to improve C4 crop yields under fluctuating environmental conditions.

plant biology↗

bioRxiv2024

Overexpression of Xanthophyll Cycle Genes Leads to Faster NPQ Acclimation in the C4 Monocot Setaria viridis

Slow relaxation and induction of Non-Photochemical Quenching (NPQ) limits plant productivity under fluctuating light in C3 plants but it is unknown if this trait extends to C4 plants. Three genes have been implicated in determining NPQ induction and relaxation rates under intermittent leaf shading; VDE and ZEP interconvert the xanthophyll pigments violaxanthin and zeaxanthin, and PsbS regulates the overall level of NPQ. Here we report the overexpression of the Arabidopsis thaliana VDE, ZEP, and PsbS in the NADP-Malic Enzyme C4 monocot Setaria viridis. We demonstrate these transgenic plants have faster NPQ induction and relaxation under fluctuating light but observe no impact on Photosystem II photochemistry yield. Under illumination, faster NPQ induction resulted in higher transient NPQ levels and a lower overall chlorophyll fluorescence yield. Together this introduces the potential for lower productivity due to over-quenching of chlorophyll during light fluctuation. Although we observed faster NPQ acclimation rates in our transgenic lines, hyperspectral imaging and pigment analysis suggest that there is little photosynthetic advantage to increasing NPQ acclimation over wildtype due to naturally fast C4 acclimation rates. These results indicate that further work is necessary in evaluating the role of NPQ acclimation in C4 growth dynamics towards the goal of improving crop productivity.

plant biology↗

bioRxiv2019

Enzyme kinetics of tobacco Rubisco expressed in Escherichia coli varies depending on the small subunit composition

AbstractRubisco catalyzes the first step in carbon fixation and has been a strategic target to improve photosynthetic efficiency. In plants, Rubisco is a complex made up of eight large subunits encoded by a chloroplast gene, rbcL, and eight small subunits expressed from a nuclear gene family and targeted to chloroplast stroma. Biogenesis of Rubisco in plants requires a chaperonin system composed of Cpn60, Cpn60{beta} and Cpn20, which helps fold the large subunit, and multiple chaperones including RbcX, Raf1, Raf2 and BSD2, which help the dimerization of the folded large subunits and subsequent assembly with the small subunits into L8S8 holoenzymes. A recent study successfully assembled functional Arabidopsis Rubisco in Escherichia coli by co-expressing the two subunits with Arabidopsis chaperonins and chaperones (Aigner et al., 2017). In this study, we modified the expression vectors used in that study and adapted them to express tobacco Rubisco by replacing the Arabidopsis genes with tobacco ones. Next, we surveyed the small subunits present in tobacco, co-expressed each with the large subunit and successfully produced active tobacco enzymes composed of different small subunits in E. coli. These enzymes produced in E. coli have carboxylation kinetics very similar to that of the native tobacco Rubisco. We also produced tobacco Rubisco with a recently discovered trichome small subunit in E. coli and found that it has a higher catalytic rate and a lower CO2 affinity compared to the enzymes with other small subunits. Our improvements in the E. coli Rubisco expression system will allow us to probe features of both the chloroplast and nuclear-encoded subunits of Rubisco that affect its catalytic rate and CO2 specificity.

plant biology↗