ARP2/3 complex associates with peroxisomes to participate in pexophagy in plants.

Actin-related protein (ARP2/3) complex is a heteroheptameric protein complex, evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane-endoplasmic reticulum contact sites. Here we demonstrate that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile spot-like structures in the cytoplasm that are associated with peroxisomes in Arabidopsis and tobacco. ARP2/3 is found at the peroxisome periphery and contains the assembled ARP2/3 complex and the WAVE/SCAR complex subunit NAP1. This ARP2/3-positive peroxisomal domain colocalizes with the autophagosome and, under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f and peroxisome-associated ARP2/3 interact in vivo with the ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than wild type, we suggest that ARP2/3 has a novel role in the process of peroxisome degradation by autophagy, called pexophagy.

CRISPR-Cas9 Arabidopsis mutants of genes for ARPC1 and ARPC3 subunits of ARP2/3 complex reveal differential roles of complex subunits.

Protein complex Arp2/3 has a conserved role in the nucleation of branched actin filaments. It is constituted of seven subunits, including actin-like subunits ARP2 and ARP3 plus five other subunits called Arp2/3 Complex Component 1 to 5, which are not related to actin. Knock-out plant mutants lacking individual plant ARP2/3 subunits have a typical phenotype of distorted trichomes, altered pavement cells shape and defects in cell adhesion. While knock-out mutant Arabidopsis plants for most ARP2/3 subunits have been characterized before, Arabidopsis plant mutants missing ARPC1 and ARPC3 subunits have not yet been described. Using CRISPR/Cas9, we generated knock-out mutants lacking ARPC1 and ARPC3 subunits. We confirmed that the loss of ARPC1 subunits results in the typical ARP2/3 mutant phenotype. However, the mutants lacking ARPC3 subunits resulted in plants with surprisingly different phenotypes. Our results suggest that plant ARP2/3 complex function in trichome shaping does not require ARPC3 subunit, while the fully assembled complex is necessary for the establishment of correct cell adhesion in the epidermis.

5-Azacytidine mediated reactivation of silenced transgenes in potato (Solanum tuberosum) at the whole plant level.

KEY MESSAGE: Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level. Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10 µM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2 days.

Arp2/3 complex subunit ARPC2 binds to microtubules.

Arp2/3 complex plays a fundamental role in the nucleation of actin filaments (AFs) in yeasts, plants, and animals. In plants, the aberrant shaping and elongation of several types of epidermal cells observed in Arp2/3 complex knockout plant mutants suggest the importance of Arp2/3-mediated actin nucleation for various morphogenetic processes. Here we show that ARPC2, a core Arp2/3 complex subunit, interacts with both actin filaments (AFs) and microtubules (MTs). Plant GFP-ARPC2 expressed in Nicotiana tabacum BY-2 cells, leaf epidermal cells of Nicotiana benthamiana and root epidermal cells of Arabidopsis thaliana decorated MTs. The interaction with MTs was demonstrated by pharmacological approach selectively interfering with either AFs or MTs dynamics as well as by the in vitro co-sedimentation assays. A putative MT-binding domain of tobacco NtARPC2 protein was identified using the co-sedimentation of several truncated NtARPC2 proteins with MTs. Newly identified MT-binding ability of ARPC2 subunit of Arp2/3 complex may represent a new molecular mechanism of AFs and MTs interaction.