Biology subjects

Pastor, B.

Publications and source records attributed to Pastor, B..

3 recordsLinked to original sources

bioRxiv2025

Investigating the role of stomatal dynamics on agronomic traits using a slac1-2 Zea mays mutant

The production of staple food crops is becoming increasingly difficult due to the amount of freshwater needed to realize food security for a growing global population. Climate projections show hotter and drier growing seasons in traditionally productive agricultural regions, which will increase the demand for limited water resources. Thus, strategies to improve water use efficiency will only become more important for sustainable agriculture. At the leaf level, stomatal conductance has a large influence on transpirational water loss and therefore water use efficiency. The anion channel SLAC1 has been shown in several plant species to function as the primary mechanism for stomatal closure, which allows stomatal aperture to change dynamically in response to environmental stimuli. Given that slac1 is a single gene that can significantly alter stomatal conductance, it has the potential to serve as a control point for improving water use efficiency. Here we fully characterize a Zea mays slac1-2 mutant in multiple field environments. Interestingly, homozygous slac1-2 hybrids did not show improved net CO2 assimilation or increased grain yield despite having greater stomatal conductance compared to a wild-type hybrid. Net CO2 assimilation and grain yield were either lower or similar in slac1-2 compared to wild-type across environments. Furthermore, the slac1-2 hybrid did not have increased nitrogen uptake. These results suggest that the C4 carbon concentrating mechanism removes any stomatal conductance limitations to CO2 assimilation, even in highly productive wild-type Z. mays hybrids. Because the slac1-2 hybrids eliminate stomatal conductance as a major variable for modulating water loss, future studies will be able to investigate alternate regulators of plant water potential to identify novel mechanisms for increasing water use efficiency.

physiology↗

bioRxiv2022

Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA

BackgroundBecause circulating DNA (cirDNA) are mainly detected as mononucleosome-associated circulating DNA (mono-N cirDNA) in blood apoptosis has until now been considered as the main source of cirDNA. The mechanism of cirDNA release into the circulation, however, is still not fully understood. This work addresses that knowledge gap, working from the postulate that neutrophil extracellular traps (NET) may be a source of cirDNA, and by investigating whether NET may directly produce mono-N cirDNA MethodsWe used the synergistic analytical information provided by specifically quantifying DNA by qPCR, and analyzing fragment size analysis by shallow WGS, and capillary electrophoresis to unequivocally study the following: the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; in vitro NET degradation in serum. We also performed an in vivo study in knockout mice, and an in vitro study of gHMW DNA degradation, to elucidate the role of NE and MPO in effecting DNA degradation and fragmentation. We then compared the NET associated markers and fragmentation size profiles of cirDNA in plasma obtained from patients with inflammatory diseases found to be associated with NET formation and high levels of cirDNA (COVID-19, N= 28; systemic lupus erythematosus, N= 10; metastatic colorectal cancer, N= 10; and from healthy individuals, N= 114). ResultsOur studies reveal that: gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24H incubation in serum corresponded to mono-N DNA); "ex vivo" NET formation, as demonstrated by a concurrent 5-, 5- and 35-fold increase of NE, MPO, and cell-free DNA (cfDNA) concentration in PMA-activated neutrophil culture supernatant, leads to the release of high molecular weight DNA that degrades down to mono-N in serum; NET mainly in the form of gHMW DNA generate mono-N cirDNA (2% and 41% of the remaining DNA after 2 hours in serum corresponded to 1-10 kbp fragments and mono-N, respectively) independent of any cellular process when degraded in serum; NE and MPO may contribute synergistically to NET autocatabolism, resulting in a 25-fold decrease in total DNA concentration and a DNA fragment size profile similar to that observed from cirDNA following 8h incubation with both NE and MPO; the cirDNA size profile of NE KO mice significantly differed from that of the WT, suggesting NE involvement in DNA degradation; and a significant increase in the levels of NE, MPO and cirDNA was detected in plasma samples from lupus, COVID-19 and mCRC, showing a high correlation with these inflammatory diseases, while no correlation of NE and MPO with cirDNA was found in HI. ConclusionsOur work thus describes the mechanisms by which NET and cirDNA are linked, by demonstrating that NET are a major source of mono-N cirDNA independent of apoptosis, and thus establishing a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions, as well as demonstrating a link between immune response and cirDNA.

molecular biology↗

bioRxiv2021

Plasma derived cell-free mitochondrial DNA originates mainly from circulating cell-free mitochondria

Circulating mitochondrial DNA (cir-mtDNA) could have a potential comparable to circulating nuclear DNA (cir-nDNA), with numerous applications. However, research and development in this area have fallen behind, particularly considering its origin and structural features. To tackle this, we initially combined Q-PCR and low-pass whole genome sequencing in the same analytical strategy previously and successfully used for cir-nDNA. This revealed unexplained structural patterns and led us to correlate these data with observations made during physical examinations such as filtration, and differential centrifugation in various plasma preparations. Both the integrity index and number of reads revealed a very minor proportion of low size-ranged fragments (<1000 bp) in plasma obtained with a standard preparation (0.06%). Filtration and high speed second step centrifugation revealed that 98.7 and 99.4% corresponded to extracellular mitochondria either free or in large extracellular vesicles. When avoiding platelet activation during plasma preparation, the proportion of both types of entities was still preponderant (76-80%), but the amount of detected mitochondrial DNA decreased 67-fold. In correlation with our previous study on the presence of circulating cell-free mitochondria in blood, our differential centrifugation procedure suggested that cir-mtDNA is also associated with approximately 18% small extracellular vesicles, 1.7% exosomes and 4% protein complexes.

genomics↗