The knock‐out of ARP3a gene affects F‐actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens

Abstract

The seven subunit Arp2/3 complex is a highly conserved nucleation factor of actin microfilaments. We have isolated the genomic sequence encoding a putative Arp3a protein of the moss Physcomitrella patens. The disruption of this ARP3A gene by allele replacement has generated loss-of-function mutants displaying a complex developmental phenotype. The loss-of function of ARP3A gene results in shortened, almost cubic chloronemal cells displaying affected tip growth and lacking differentiation to caulonemal cells. In moss arp3a mutants, buds differentiate directly from chloronemata to form stunted leafy shoots having differentiated leaves similar to wild type. Yet, rhizoids never differentiate from stem epidermal cells. To characterize the F-actin organization in the arp3a-mutated cells, we disrupted ARP3A gene in the previously described HGT1 strain expressing conditionally the GFP-talin marker. In vivo observation of the F-actin cytoskeleton during P. patens development demonstrated that loss-of-function of Arp3a is associated with the disappearance of specific F-actin cortical structures associated with the establishment of localized cellular growth domains. Finally, we show that constitutive expression of the P. patens Arp3a and its Arabidopsis thaliana orthologs efficiently complement the mutated phenotype indicating a high degree of evolutionary conservation of the Arp3 function in land plants.

Actin Interacting Protein1 and Actin Depolymerizing Factor Drive Rapid Actin Dynamics inPhyscomitrella patens

“…The AIP1 knockout phenotype is most reminiscent of Arp2/3 complex mutants in moss, which are also viable and have reduced tip growth. However, the Arp2/3 complex mutants display more penetrant defects with complete inhibition in the formation of caulonemal cells and rhizoids and mild to severe defects in gametophore development (Harries et al, 2005;Perroud and Quatrano, 2006;Finka et al, 2008). It is interesting that AIP1 (a depolymerizer with ADF) and the Arp2/3 complex (a nucleator) share a similar phenotype.…”

“…Indeed genetic studies support the requirement for a number of actin binding proteins in the expansion of tip-growing cells . These include actin-nucleating proteins, such as formins (Vidali et al, 2009c;Ye et al, 2009;Cheung et al, 2010) and the Arp2/3 complex (Harries et al, 2005;Perroud and Quatrano, 2006;Finka et al, 2008), the filament-stabilizing fimbrins , actin monomer binding proteins like profilin (Ramachandran et al, 2000;Vidali et al, 2007Vidali et al, , 2009b, and actin turnover proteins like actin depolymerizing factor (ADF) (Augustine et al, 2008;Vidali et al, 2009b), villin Zhang et al, 2011), and actin interacting protein1 (AIP1) (Ketelaar et al, 2004). It is tempting to suggest that these actin binding proteins are important for tip growth by promoting the reorganization of F-actin.…”

Plasma Membrane Cyclic Nucleotide Gated Calcium Channels Control Land Plant Thermal Sensing and Acquired Thermotolerance

“…Resulting products were first subcloned in pGEM-T-easy vector (Promega) and digested with ApaI-Xho or SpeI-SacII and subcloned in two steps in the corresponding sites of pBSMDII (Finka et al, 2008) to generate the pCNGCb-KO vector. To minimize the possibility of superintegration of CNGCb-KO cassette into HSP-GUS locus, ;10 mg of CNGCb-KO transgene was amplified by PCR employing standard T3 and T7 primers followed by enzymatic digestion with ApaI and SacII.…”

Can mosses serve as model organisms for forest research?

“…As moss protonema filaments elongate by tip growth, as root hairs and pollen tubes in flowering plants, the role of the cytoskeleton and associated factors has been investigated intensively (Finka et al 2008;Furt et al 2013;Vidali and Bezanilla 2012). The function of different types of myosin motor proteins were recently analyzed, confirming their conserved function in growth, organelle shape, and actin organization (Madison and Nebenführ 2013), but also identifying a novel role in cell division (Wu and Bezanilla 2014).…”