Genetius

The study characterizes the chloroplast‑localized protein AT4G33780 in Arabidopsis thaliana using CRISPR/Cas9 knockout and overexpression lines, revealing tissue‑specific expression and context‑dependent effects on seed germination, seedling growth, vegetative development, and root responses to nickel stress. Integrated transcriptomic (RNA‑seq) and untargeted metabolomic analyses show extensive transcriptional reprogramming—especially of cell‑wall genes—and altered central energy metabolism, indicating AT4G33780 coordinates metabolic state with developmental regulation rather than controlling single pathways.

The study reveals that the Arabidopsis O-GlcNAc transferase SEC is essential for timely ABA‑induced stomatal closure and drought tolerance, with sec-5 mutants showing delayed closure and increased water loss, while SEC overexpression enhances responsiveness. SEC influences guard‑cell microtubule remodeling, as loss of SEC impairs microtubule reorganization and SEC directly interacts with tubulin α‑4, suggesting tubulin as a target of O‑GlcNAcylation.

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.

The study investigated the five Arabidopsis SCAMP proteins, focusing on SCAMP5, and identified conserved tyrosine and NPF motifs that mediate anterograde transport and endocytosis, respectively. SCAMPs were shown to dimerize at the plasma membrane and endosomes, interact with plasma‑membrane aquaporins, and their loss (triple and quintuple mutants) conferred mild developmental delay but increased drought tolerance, likely via altered PIP trafficking or stability.

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.

Loss‑of‑function mutations in the drought‑induced genes GASA3 and AFP1 confer enhanced drought tolerance in Arabidopsis thaliana, primarily through smaller stomatal apertures and increased ABA accumulation via hydrolysis of ABA‑GE. Constitutive overexpression of these genes heightens drought sensitivity, indicating that the AFP1/GASA3 module negatively regulates stomatal closure and ABA signaling.

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.