Biology subjects

Puchta-Jasinska, M.

Publications and source records attributed to Puchta-Jasinska, M..

3 recordsLinked to original sources

bioRxiv2025

New molecular players: siRNA expression and gene regulation in Hordeum vulgare ageing seeds after germination

Small interfering RNAs (siRNAs), a subclass of small non-coding RNAs, play crucial roles in regulating seed germination and viability through epigenetic mechanisms like RNA-directed DNA methylation (RdDM). This study presents the first comprehensive investigation of siRNA profiles linked to seed viability and germination in barley (Hordeum vulgare), utilizing a unique set of seeds from a single batch subjected to controlled long-term storage. Some seeds lost viability due to moisture exposure from unsealing, creating a natural experimental model to explore vigor effects. sRNA sequencing revealed 85,728 differentially expressed siRNAs, with distinct patterns between regenerated, high-viability, and low-viability seeds. Notably, trans-acting siRNAs (ta-siRNAs) showed peak abundance at different imbibition times depending on seed quality, suggesting dynamic regulation. Around 46% of siRNAs were 21 nucleotides, and 54% were 22 nucleotides long. Gene Ontology and degradome analyses confirmed siRNA target genes involved in vital biological processes such as cytochrome complex function, root development, cell maturation, and carbohydrate metabolism. Despite RNA degradation in low-viability seeds, siRNAs remained relatively stable, indicating their potential role in maintaining seed metabolic activity during dormancy release and germination initiation. This pioneering research uncovers novel insights into siRNA-mediated control of seed longevity and germination, highlighting the innovative use of stable, well-characterized plant material to disentangle molecular mechanisms underpinning seed vigor and germination success.

plant biology↗

bioRxiv2024

Improved Degradome Sequencing Protocol via Reagent Recycling from sRNAseq Library Preparations

BackgroundOne of the key elements in the analysis of gene expression and its post-translational regulation is miRNAs. Degradome-seq analyses are performed to analyze the cleavage of target RNAs in the transcriptome. In this work, an improved library preparation protocol for degradome sequencing is presented. The developed protocol improves the efficiency of library preparation in degradome-seq analysis used to identify microRNA targets, reduces the time of library preparation and lowers the cost of purchasing reagents.. ResultsThe aim of this study was the development of an efficient protocol for the construction of degradome sequencing libraries using residual reagents from the sRNA-seq library preparation kit. To this end, modified primers and adaptors were designed. The library purification step based on automated electrophoresis and high-resolution agarose was modified and optimized in the presented protocol. Size standards of 60 and 65 bp were developed. They were prepared for precise band excision from the gel. Cloning to plasmid and sequencing of the inserted fragment, i.e., a fragment from the degradome library, verified the correctness of the library preparation using the developed protocol. ConclusionThe developed protocol allowed the construction and sequencing of degradome libraries even from RNA samples with low RIN. It significantly reduces the cost of library construction. This is due to the use of residues from the sRNA-seq library kit. The precision of the excised fragment after electrophoresis performed during the procedure to isolate fragments of the correct length is significantly improved by the use of additional size markers. Compared to previously used methods, optimizing the purification method of degradom-seq libraries allowed to increase the yield of fragments obtained. Notably, the time required for the entire library preparation protocol does not exceed three days, also a significant time savings.

plant biology↗

bioRxiv2024

Aging and Germination of Long-term Stored Seeds: Can MicroRNAs Unlock the Secrets?

BackgroundSmall non-coding RNAs appear to be one of the key components of the germination process. To investigate how small non-coding RNAs correlate with germination of seeds with different levels of viability, miRNA-Seq analyses were performed. ResultsOur analysis sequencing identified 62 known miRNAs from 11 families and 234 new miRNAs after imbibition process. Among the miRNAs with the highest expression levels, we can mention: miR159, miR168 and miR166. The study placed particular emphasis on miRNAs with significant differences in expression levels at different stages of imbibition and among seeds with different viability. DEG analysis identified 28 miRNAs with significant differences in expression levels, their function was assessed by in silico analyses and confirmed by degradome-seq analysis. The expression of miRNAs was verified by qRT-PCR. ConclusionOur data provides a useful source of information on miRNA during germination long term storage seeds with different viability. The studies suggest that miRNAs are involved in the germination process by their regulation DNA and RNA binding, regulation of developmental process and ribosome.

plant biology↗