Profilin Is Essential for Tip Growth in the MossPhyscomitrella patens

Abstract

The actin cytoskeleton is critical for tip growth in plants. Profilin is the main monomer actin binding protein in plant cells. The moss Physcomitrella patens has three profilin genes, which are monophyletic, suggesting a single ancestor for plant profilins. Here, we used RNA interference (RNAi) to determine the loss-of-function phenotype of profilin. Reduction of profilin leads to a complete loss of tip growth and a partial inhibition of cell division, resulting in plants with small rounded cells and fewer cells. We silenced all profilins by targeting their 39 untranslated region sequences, enabling complementation analyses by expression of profilin coding sequences. We show that any moss or a lily (Lilium longiflorum) profilin support tip growth. Profilin with a mutation in its actin binding site is unable to rescue profilin RNAi, while a mutation in the poly-L-proline binding site weakly rescues. We show that moss tip growing cells contain a prominent subapical cortical F-actin structure composed of parallel actin cables. Cells lacking profilin lose this structure; instead, their F-actin is disorganized and forms polarized cortical patches. Plants expressing the actin and poly-L-proline binding mutants exhibited similar F-actin disorganization. These results demonstrate that profilin and its binding to actin are essential for tip growth. Additionally, profilin is not needed for formation of F-actin, but profilin and its interactions with actin and poly-L-proline ligands are required to properly organize F-actin.

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

“…Other essential proteins critically required for tip growth, such as profilin (Vidali et al, 2007), class II formin (Vidali et al, 2009c), and myosin XI (Vidali et al, 2010), are present in small gene families. Since moss is predominately haploid, having multiple redundant copies of essential genes may be a safeguard in the event of mutation of one of the gene copies or a way to ensure sufficient expression levels.…”

“…To amplify the AIP1 59 and AIP1 39 regions, wild-type genomic DNA was used as a template for PCR amplification with primers (see Supplemental Table 2 online). The resistance cassette was generated by amplifying a fragment containing the NOS terminator and hygromycin resistance cassette from pTHUBIGate (Vidali et al, 2007). All primers (see Supplemental Table 2 online) contained the appropriate attB sites, and BP clonase reactions were performed to introduce the PCR products into their respective pDONR vectors to generate the entry clones L1L4-SwaI-AIP-59, R4R3-NOSterLox-Hygro-Lox, and L3L2-AIP-39-SwaI.…”

“…To generate expression constructs, LR clonase reactions (Invitrogen) recombined the pENT-AIP1 and pENT-ADF (Augustine et al, 2008) clones into the pTH-UbiGate (Vidali et al, 2007) and pMK-UbiGate destination vectors. pMK-UbiGate was generated by modifying pMBL5 (Bezanilla et al, 2003) to include the maize (Zea mays) ubiquitin promoter, Gateway cassette, and NOS terminator.…”

“…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.…”

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

“…Abolition of tip growth in protonemal cells was the most visible common phenotype observed after deletion of ARPC4 [Perroud and Quatrano, 2006], of BRICK1 (a member of the Scar/Wave complex ) or after knock-down (KD) of ARPC1 [Harries et al, 2005], of profilin, PRF-KD [Vidali et al, 2007] or of actin depolymerization factor, ADF-KD [Augustine et al, 2008]. Yet, differences were also observed between these mutants: PRF-KD and ADF-KD are dramatically affected in both cell growth and division processes and rapidly dies, whereas the other mutants are viable, and almost normal gametophores differentiate in ARPC4-KO and BRICK1-KO but not in ARPC1-KD.…”