A transcription factor WRKY36 interacts with AFP2 to break primary seed dormancy by progressively silencing DOG1 in Arabidopsis.

The phytohormones abscisic acid (ABA) and gibberellic acid (GA) antagonistically control the shift between seed dormancy and its alleviation. DELAY OF GERMINATION1 (DOG1) is a critical regulator that determines the intensity of primary seed dormancy, but its underlying regulatory mechanism is unclear. In this study, we combined physiological, biochemical, and genetic approaches to reveal that a bHLH transcriptional factor WRKY36 progressively silenced DOG1 expression to break seed dormancy through ABI5-BINDING PROTEIN 2 (AFP2) as the negative regulator of ABA signal. AFP2 interacted with WRKY36, which recognizes the W-BOX in the DOG1 promoter to suppress its expression; Overexpressing WRKY36 broke primary seed dormancy, whereas wrky36 mutants showed strong primary seed dormancy. In addition, AFP2 recruited the transcriptional corepressor TOPLESS-RELATED PROTEIN2 (TPR2) to reduce histone acetylation at the DOG1 locus, ultimately mediating WRKY36-dependent inhibition of DOG1 expression to break primary seed dormancy. Our result proposes that the WRKY36-AFP2-TPR2 module progressively silences DOG1 expression epigenetically, thereby fine-tuning primary seed dormancy.

Computer-aided design of reversible hybridization chain reaction (CAD-HCR) enables multiplexed single-cell spatial proteomics imaging.

In situ spatial proteomics analysis of a single cell has not been achieved yet, mainly because of insufficient throughput and sensitivity of current techniques. Recent progress on immuno-nucleic acid amplification technology presents tremendous opportunities to address this issue. Here, we report an innovative hybridization chain reaction (HCR) technique that involves computer-aided design (CAD) and reversible assembly. CAD enables highly multiplexed HCR with a sequence database that can work in parallel, while reversible assembly enables the switching of HCR between a working state and a resting state. Thus, CAD-HCR has been successfully adopted for single-cell spatial proteomics analysis. The fluorescence signal of CAD-HCR is comparable with conventional immunofluorescence, and it is positively correlated with the abundance of target proteins, which is beneficial for the visualization of proteins. The method developed here expands the toolbox of single-cell analysis and proteomics studies, as well as the performance and application of HCR.

Nondestructive analysis of tumor-associated membrane protein MUC1 in living cells based on dual-terminal amplification of a DNA ternary complex.

Non-destructive analysis of cells at the molecular level is of critical importance for cell research. At present, immunoassay-based and aptamer-based methods can achieve non-structural destructive cell analysis, but still lead to changes in cells at the molecular level. Here, we have proposed a dual-terminal amplification (DTA) strategy, which enables nondestructive analysis of membrane protein MUC1 without the effect on protein expression and cell viability in living cells. Methods: A fluorophore (Cy5)-labeled DNA ternary complex consisting of three oligonucleotides is designed. It can recognize MUC1 through its aptamer region, and thus make the MUC1 of cells visible under a fluorescence microscope. When DNA polymerase is added, dual-terminal amplification is performed. One direction dissociates aptamer from MUC1, and the other direction, also known as rolling circle amplification (RCA), produces long linear DNA strands, which can be further adopted for quantitative analysis of MUC1. In this way, all reagents are removed from the surface of the cells after the analysis, which allows nondestructive analysis. We named this strategy dual-terminal amplification (DTA) analysis. Results: By using the DTA analysis, both in situ fluorescence imaging analysis and ex situ fluorescence quantitative analysis of MUC1 were achieved. In addition, the aptamer-containing DNA ternary complex stays on cell surface only during the analysis and leaves the cell after the analysis is complete. The cells can be maintained in a non-interfering state for the rest of the time. So after the analysis, it is found that there are no effect on the physiological activity of cells and the expression of target protein even after two rounds of repeatable imaging and quantitative analysis. Conclusion: In summary, we have successfully constructed a strategy for nondestructive analysis of membrane protein in living cells. We believe that this method provides a promising way for the analysis of the key membrane proteins of cells and the versatile utilization of precious cell samples.