Arabidopsis FH1 Formin Affects Cotyledon Pavement Cell Shape by Modulating Cytoskeleton Dynamics

Abstract

Plant cell morphogenesis involves concerted rearrangements of microtubules and actin microfilaments. We previously reported that FH1, the main Arabidopsis thaliana housekeeping Class I membrane-anchored formin, contributes to actin dynamics and microtubule stability in rhizodermis cells. Here we examine the effects of mutations affecting FH1 (At3g25500) on cell morphogenesis and above-ground organ development in seedlings, as well as on cytoskeletal organization and dynamics, using a combination of confocal and variable angle epifluorescence microscopy with a pharmacological approach. Homozygous fh1 mutants exhibited cotyledon epinasty and had larger cotyledon pavement cells with more pronounced lobes than the wild type. The pavement cell shape alterations were enhanced by expression of the fluorescent microtubule marker GFP-microtubule-associated protein 4 (MAP4). Mutant cotyledon pavement cells exhibited reduced density and increased stability of microfilament bundles, as well as enhanced dynamics of microtubules. Analogous results were also obtained upon treatments with the formin inhibitor SMIFH2 (small molecule inhibitor of formin homology 2 domains). Pavement cell shape in wild-type (wt) and fh1 plants in some situations exhibited a differential response towards anti-cytoskeletal drugs, especially the microtubule disruptor oryzalin. Our observations indicate that FH1 participates in the control of microtubule dynamics, possibly via its effects on actin, subsequently influencing cell morphogenesis and macroscopic organ development.

A new kymogram-based method reveals unexpected effects of marker protein expression and spatial anisotropy of cytoskeletal dynamics in plant cell cortex

“…Kymograms are sometimes used to qualitatively demonstrate plant cytoskeletal dynamics (e.g. [22–26]).…”

“…Nevertheless, quantitative analysis of cytoskeletal dynamics remains challenging, especially in the case of rapidly changing and often weakly labeled actin structures, and techniques involving manual tracking or counting of individual filaments or filament bundles thus remain the method of choice (e.g. [23, 25, 26, 33, 34]). Such a manual approach is usually very laborious and may be prone to observer bias (which can be eliminated by a blinded study design involving an evaluator who does not know which sample is which).…”

On growth and formins

“…24 We recently reported that impairment of formin function in Arabidopsis (either by mutations in the FH1 locus or by pharmacological intervention) leads to increased complexity of cotyledon pavement cell shape, associated with stabilization and bundling of microfilaments, reduced density of fine cortical actin meshwork, and increased dynamics of cortical microtubules. 25 Thus, formins, members of an evolutionarily ancient family of eukaryotic proteins possessing the FH2 domain whose dimer can nucleate actin, may play a key part in such actin-microtubule co-ordination. Indeed, formins often participate in microtubule organization in both metazoans and plants (reviewed in refs.…”

“…In Arabidopsis, FH1 is the most ubiquitously expressed Class I formin during vegetative development (see refs. 25,27). This may explain the similarity of pavement cell shape changes caused by fh1 loss-of-function mutation and by general inhibition of formin-dependent actin polymerization by treatment with the SMIFH2 compound.…”

“…Membrane-anchored formins may contribute to the focusing of microtubule bundles to future indentations by restricting lateral mobility of cortical microtubules through nucleation of membrane-attached microfilaments, promoting assembly of cortically anchored actin cables. 25 Unlike ARP2/3-nucleated actin arrays, these cables might form only a sparse meshwork, sufficient to prevent microtubule bundling but not interfering with exocytosis (compare ref. 21).…”