Primer pairs used for genotyping.

<div><p>Protein phosphatase 2AB’’ (PP2A B’’) family subunits have calcium-binding EF-hand motifs, facilitating interaction with PP2A substrates. In <i>Arabidopsis thaliana</i>, the PP2A B’’ family subunits consist of six members, AtB’’α-ε and FASS. These subunits can interact with a basic leucine zipper transcription factor, VIP1, and its close homologs. Mechanical stress triggers PP2A-mediated dephosphorylation of VIP1 and its close homologs, leading to nuclear localization and gene upregulation to alleviate touch-induced root bending and leaf damage. However, the physiological roles of PP2A B’’ family subunits in the mechanical stress response in Arabidopsis remain unclear. This study aims to characterize such roles. A quadruple knockout mutant with T-DNA insertions in <i>AtB’’α</i>, <i>AtB’’β</i>, <i>AtB’’γ</i>, and <i>AtB’’δ</i> was generated. <i>atb’’αβγδ</i> mutants exhibited no significant damage upon brushing or touch-induced root bending compared to the wild type. Transcriptome analysis showed a significant decrease in the expression of <i>CYP707A3</i>, a gene potentially targeted by VIP1 that regulates abscisic acid (ABA) catabolism, in the <i>atb’’αβγδ</i> mutant compared to wild type leaves. However, other genes, including <i>XTH23</i>, <i>EXLA1</i>, and <i>CYP707A1</i>, also VIP1 targets, exhibited similar induction in both brushed <i>atb’’αβγδ</i> mutants and wild type leaves. We observed an enrichment of the CAMTA motif, CGCG(C/T) in the promoters of genes showing downregulated expression levels in brushed <i>atb’’αβγδ</i> leaves compared to brushed wild type leaves. These findings suggest that PP2A B’’ family subunits exhibit functional redundancy in the VIP1-dependent pathway but influence CAMTA-dependent gene expression under mechanical stress. Under calcium-deficient and ABA-supplemented conditions, growth of <i>atb’’αβγδ</i> seedlings was retarded when compared to wild type and single knockout mutants, <i>atb’’γ</i> and <i>atb’’δ</i>, indicating a crucial role in plant development by modulating calcium or ABA signaling.</p></div>