Grant, C. A. | bioRxiv Science

bioRxiv2022

PIF4/HEMERA-mediated daytime thermosensory growth requires the Mediator subunit MED14

While moderately elevated ambient temperatures do not trigger stress responses in plants, they do significantly stimulate the growth of specific organs through a process known as thermomorphogenesis. The basic helix-loop-helix transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) plays a central role in regulating thermomorphogenetic hypocotyl elongation in various plant species, including Arabidopsis thaliana. Although it is well known that PIF4 promotes plant thermosensory growth by activating genes involved in the biosynthesis and signaling of the phytohormone auxin, the detailed molecular mechanism of such transcriptional activation is not clear. Our previous studies demonstrated that HEMERA (HMR), a transcriptional co-activator of PIF4, promotes the thermo-induced expression of PIF4 target genes through its nine-amino-acid transactivation domain (9aaTAD). In this report, we investigate the role of the Mediator complex in the PIF4/HMR-mediated thermoresponsive gene expression. Through the characterization of various mutants of the Mediator complex, a tail subunit named MED14 is identified as an essential factor for thermomorphogenetic hypocotyl growth. MED14 is required for the thermal induction of PIF4 target genes but has a marginal effect on the levels of PIF4 and HMR. Further transcriptomic analyses confirm that the expression of numerous PIF4/HMR-dependent, auxin-related genes requires MED14 at warm temperatures. Moreover, PIF4 and HMR physically interact with MED14 and both are indispensable for the association of MED14 with the promoters of these thermoresponsive genes. Taken together, these results unveil an important thermomorphogenetic mechanism, in which PIF4 and HMR recruit the Mediator complex to activate auxin-related growth-promoting genes when plants sense moderate increases in ambient temperature. One-sentence summaryThe Mediator subunit MED14 promotes thermomorphogenesis by interacting with PHYTOCHROME-INTERACTING FACTOR 4 and HEMERA to induce the expression of growth-promoting genes at elevated temperatures.

plant biology↗

bioRxiv2020

CelLEVITAS: Label-free, rapid sorting and enrichment of live cells via magnetic levitation

Sorting methods that remove non-viable cells and debris, while retaining a high yield of viable cells, are crucial for many applications in biotechnology, genomics, tissue engineering and medicine. However, a significant challenge is gentle sorting of these different cell states based on very minute differences in density and magnetic signatures, without relying on any labels, tags or markers. Here, a new magnetic levitation-based technology, CelLEVITAS, is developed for the label-free sorting and enrichment of live cells. This work reports the first use of magnetic levitation for sorting of viable and non-viable cells within a microfluidic device and demonstrates extremely effective removal of dead cells and debris from heterogeneous samples. First, the levitation conditions for separating viable and non-viable cells under a magnetic field were fine-tuned. Levitation trajectories of live and dead cell states were then monitored in real-time, as cells magnetically focused to their corresponding levitation bands. CelLEVITAS successfully sorted and enriched live cells from a variety of input cell concentrations (100-200,000 cells/mL) and a variety of input purities (10-50%) into consistently high output purities (>80%). This method is sensitive, does not impair cell viability during sorting, and significantly increases the input sample viability up to 7-fold. Overall, this new magnetic levitation-based sorting strategy drastically reduces the processing time to a single-step, 30-minute sorting protocol and eliminates the manual pre-processing and labeling steps that are required for traditional flow cytometry techniques.

bioengineering↗