MERISTEM-DEFECTIVE / DEFECTIVELY ORGANIZED TRIBUTARIES2 regulates the balance between stemness and differentiation in the root meristem through RNA splicing control

Abstract

Plants respond to environmental stresses through controlled stem cell maintenance and meristem activity. One level of transcriptional control is RNA alternative splicing. However the mechanistic link between stress, meristem function and RNA splicing is poorly understood. The MERISTEM-DEFECTIVE (MDF)/DEFECTIVELY ORGANIZED TRIBUTARIES (DOT2) gene of Arabidopsis encodes a SR-related family protein, required for meristem function and leaf vascularization, and is the likely orthologue of the human SART1 and yeast snu66 splicing factors. MDF is required for the correct splicing and expression of key transcripts associated with root meristem function. We identified RSZ33 and ACC1, both known to regulate cell patterning, as splicing targets required for MDF function in the meristem. MDF expression is modulated by osmotic and cold stress, associated with differential splicing and specific isoform accumulation and shuttling between nucleus and cytosol, and acts in part via a splicing target SR34. We propose a model in which MDF controls splicing in the root meristem to promote stemness and repress stress response and cell differentiation pathways.

MDF regulates both auxin-dependent and -independent pathways of adventitious root regeneration inArabidopsis

“…The MDF gene of Arabidopsis is required for root meristem organization and maintenance (Casson et al, 2009). It encodes a putative RS domain protein and has recently been demonstrated to be a splicing factor that regulates meristem function through both auxin-dependent and auxin-independent mechanisms, to maintain stemness (Thompson et al, 2023). The mutant shows decreased levels of PIN family mRNAs and this is associated with a reduced auxin maximum in the basal region of the mdf embryo and seedling root meristem.…”

POLARIS is a copper-binding peptide required for ethylene signalling control in Arabidopsis.

“…RNA was extracted from 7 day-old seedlings grown on half strength MS10 medium using the Sigma-Aldrich Plant Total RNA Kit (STRN50) and the On-Column DNase I Digestion Set (DNASE10-1SET), essentially as described ( 51 ). Tissue was ground in liquid nitrogen before incubation in lysis solution containing 2-mercaptoethanol at 65°C for 3 min.…”

“…The Illumina HiSeq 2500 System was used for RNA sequencing of three biological replicate samples, with libraries prepared using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Plant Sample Preparation kit (RS-122-2401), essentially as described ( 51 ). Ribosomal RNA (rRNA) was removed and purified RNA was quality checked using a TapeStation 2200 (Agilent Technology) with High Sensitivity RNA ScreenTape (5067-5579).…”

“…For RT-qPCR, RNA was extracted from 7 day-old seedlings (3 biological replicates, 20 mg of tissue) as described ( 51 ). Total mRNA was extracted using Dynabeads ® mRNA DIRECT™ kit with Oligo(dT) 25 labelled magnetic beads.…”

“…The Illumina HiSeq 2500 System was used for RNA sequencing of three biological replicate samples, with libraries prepared using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Plant Sample Preparation kit (RS-122-2401), essentially as described (51).…”