Genetius

The study used CRISPR/Cas9 to edit the downstream region of the Arabidopsis thaliana FLOWERING LOCUS T (FT) gene, identifying a 2.3‑kb segment containing the Block E enhancer as crucial for normal FT expression and flowering. Fine‑scale deletions pinpointed a 63‑bp core module with CCAAT‑ and G‑boxes, and revealed a cryptic CCAAT‑box that becomes active when repositioned, highlighting the importance of local chromatin context and motif arrangement for enhancer function.

Large-scale bioinformatics identified a new class of transmembrane phosphotransfer proteins (TM‑HPt) across 61 plant species, showing conserved HPt motifs and potential activity in multistep phosphorelay signaling. Phylogenetic relationships were inferred via Bayesian DNA analysis, expression was validated by transcriptomics, and molecular modeling suggested possible membrane-associated structural arrangements.

The study reconstructed the evolutionary history of plant-specific GBF1-type ARF-GEFs by building phylogenetic trees and ortho‑synteny groups, identifying orthologs of AtGNOM and AtGNL1 across species. Functional analyses using transgenic Arabidopsis lines and yeast two‑hybrid assays revealed how duplication and loss events diversified GNOM paralogs, separating polar recycling from secretory trafficking functions.

The study introduces a CRISPR/Cas9‑based restoration system (CiRBS) that reactivates a disabled luciferase reporter (LUC40Ins26bp) in transgenic Arabidopsis, enabling long‑term single‑cell bioluminescence monitoring. Restoration occurs within 24 h after particle‑bombardment‑mediated CRISPR delivery, with ~7 % of cells regaining luminescence and most restored cells carrying a single correctly edited chromosome, facilitating reliable analysis of cellular gene‑expression heterogeneity.

The study applied a CRISPR/Cas9 multiplex guide RNA strategy to delete entire open reading frames of four reproductive genes in Arabidopsis thaliana, achieving homozygous deletions already in the T1 generation with rates of 8.3–30%. Deletion efficiencies correlated with DeepSpCas9 prediction scores, and phenotypic analyses revealed unexpected effects of residual gene fragments on fertilization and seed development.