Jardine, K. | Search bioRxiv

bioRxiv2023

Concurrent measurement of O2 and isoprene production during photosynthesis: pros, cons, and metabolic implications

During oxygenic photosynthesis, oxygen (O2) is generated from water photolysis, which provides reducing power to sustain CO2 assimilation. To date, traditional leaf gas-exchange experiments have been focused on net CO2 exchange (Anet), with limited observations of net oxygen production (NOP). Here, we present the first gas-exchange/fluorescence system, coupling CO2/H2O analysis (photosynthesis and transpiration) with NOP and isoprene emission measurements. This configuration allowed us to calculate the assimilatory quotient (AQ = Anet/NOP) and thus obtain a more complete picture of the photosynthetic redox budget via photosynthetic production of O2, electron transport rate (ETR), and isoprene biosynthesis. We used cottonwood leaves (Populus trichocarpa) and carried out response curves to light, CO2 and temperature along with 18O-labelling with 18O-enriched water. We found that Anet and NOP were linearly correlated across environmental variables with AQ of 1.27 +/- 0.12 regardless of light, CO2, and temperature. Anet and NOP had optimal temperatures (Topt) of 31{degrees}C, while ETR (35{degrees}C) and isoprene emissions (39{degrees}C) had distinctly higher Topt. Leaves labelled with H218O produced labeled (18O16O) oxygen with the same Topt as ETR (35{degrees}C). The results confirm a tight connection between water oxidation and ETR and are consistent with a suppression of Anet and NOP at high temperature driven by an acceleration of (photo)respiration. The findings support the view of isoprene biosynthesis primarily driven by excess photosynthetic ATP/NADPH not consumed by the Calvin cycle during photorespiratory conditions as an important thermotolerance mechanism linked with high rates of CO2 and O2 recycling. KeywordsPhotosynthesis, net oxygen production, gross oxygen production, H218O labeling One sentence summaryA leaf gas-exchange system is presented enabling a more complete picture of the photosynthetic redox budget and calculation of the assimilatory quotient.

plant biology↗

bioRxiv2020

Rac guanine nucleotide exchange factors promoting Lgl1 phosphorylation

The protein Lgl1 has key roles in the regulation of cell polarity. We have shown that Lgl1 is inactivated by hyperphosphorylation in glioblastoma as a consequence of PTEN loss and aberrant activation of the PI 3-kinase pathway; this contributes to glioblastoma pathogenesis both by promoting invasion and repressing glioblastoma cell differentiation. Lgl1 is phosphorylated by atypical protein kinase C in a complex with Par6 and activated Rac. Here we have investigated the role of specific Rac guanine nucleotide exchange factors in Lgl1 hyperphosphorylation in glioblastoma. We used CRISPR/Cas9 to knockout PREX1, a PI 3-kinase pathway-responsive Rac guanine nucleotide exchange factor, in patient-derived glioblastoma cells. Knockout cells had reduced Lgl1 phosphorylation which could be reversed by re-expressing PREX1. PREX1 knockout cells showed reduced motility and an altered phenotype suggestive of partial neuronal differentiation; consistent with this, RNA-seq analyses identified sets of PREX1-regulated genes associated with changes in cell motility and neuronal differentiation. PREX1 knockout in glioblastoma cells from a second patient did not affect Lgl1 phosphorylation. These cells overexpressed a short isoform of the Rac guanine nucleotide exchange factor TIAM1; knockdown of TIAM1 in PREX1-knockout cells from this patient reduced Lgl1 phosphorylation. These data show that PREX1 links aberrant PI 3-kinase signaling to Lgl1 phosphorylation in glioblastoma, but that TIAM1 can also promote Lgl phosphorylation in a subset of patients. While this shows redundant mechanisms for Lgl1 phosphorylation, PREX1 appears to have a non-redundant role in glioblastoma cell motility, as this was impaired in PREX1 knockout cells from both patients.

biochemistry↗