František Baluška

Actin: A Dynamic Framework For Multiple Plant Cell Functions

Recent progress in understanding the plant actin gene family is reviewed, focusing on the Arab idopsis actins. Taking an evolutionary perspective, we have focused on the functional significance of the conserved but ancient vegetative and reproductive actin classes, which date back to the origin of vascular plants. We propose that the conservation of ancient family members is due to differential gene regulation and/or to functional differences among isovariants. The eight functional actin genes are widely dispersed on four of the five Arabidopsis chromosomes. Each of the five actin gene subclasses are strongly expressed at some time and place during plant development, and they are highly differentially regulated. A handful of surface epitope differences among plant and vertebrate actins enabled the isolation of general and subclass-specific anti-plant actin monoclonal antisera. These reagents give an excellent resolution to the switch from vegetative to reproductive actin protein expression during floral development. Combined with refined fixation protocols, these reagents resolve the intimate relationship between the chloroplasts and the actin cytoskeleton in leaf cells. Sequence-based screening procedures were developed for the isolation of the first mutant alleles of plant actins. These mutants have strong deleterious effects on the survival of plants and are effectively lethal mutations over several generations. Sequence differences among the co-expressed plant actin isovariants should produce complex dynamics within actin filaments and with actin-binding proteins. Future work on the significance of this ancient family will focus on the cell biology, genetics, and biochemistry of the isovariants.

F-actin-dependent endocytosis of cell wall pectins in meristematic root cells: insights from Brefeldin A-induced compartments

Brefeldin A (BFA) inhibits exocytosis but allows endocytosis, making it a valuable agent to identify molecules that recycle at cell peripheries. In plants, formation of large intracellular compartments in response to BFA treatment is a unique feature of some, but not all, cells. Here, we have analyzed assembly and distribution of BFA compartments in development- and tissue-specific contexts of growing maize (Zea mays) root apices. Surprisingly, these unique compartments formed only in meristematic cells of the root body. On the other hand, BFA compartments were absent from secretory cells of root cap periphery, metaxylem cells, and most elongating cells, all of which are active in exocytosis. We report that cell wall pectin epitopes counting rhamnogalacturonan II dimers cross-linked by borate diol diester, partially esterified (up to 40%) homogalacturonan pectins, and (1→4)-β-d-galactan side chains of rhamnogalacturonan I were internalized into BFA compartments. In contrast, Golgi-derived secretory (esterified up to 80%) homogalacturonan pectins localized to the cytoplasm in control cells and did not accumulate within characteristic BFA compartments. Latrunculin B-mediated depolymerization of F-actin inhibited internalization and accumulation of cell wall pectins within intracellular BFA compartments. Importantly, cold treatment and protoplasting prevented internalization of wall pectins into root cells upon BFA treatment. These observations suggest that cell wall pectins of meristematic maize root cells undergo rapid endocytosis in an F-actin-dependent manner.

Endocytosis, Actin Cytoskeleton, and Signaling

Endocytosis is the internalization of plasma membrane proteins and lipids, extracellular molecules, fluids, particles, exosomes, viruses, and bacteria. Endocytic internalization is a conserved process for all eukaryotic cells that is required for diverse cellular functions. These include turnover

Cytoskeleton-Plasma Membrane-Cell Wall Continuum in Plants. Emerging Links Revisited

Eukaryotic cells typically deviate from spherical shapes due to complex interactions between elements of their cytoskeleton and the extracellular matrix (ECM). Communication between the cytoskeleton and ECM is one of the most characteristic features of cellular mechanics and allows cells to respond

Aluminum-Induced Gene Expression and Protein Localization of a Cell Wall-Associated Receptor Kinase in Arabidopsis

Here, we report the aluminum (Al)-induced organ-specific expression of a WAK1 (cell wall-associated receptor kinase 1) gene and cell type-specific localization of WAK proteins in Arabidopsis. WAK1-specific reverse transcriptase-polymerase chain reaction analysis revealed an Al-induced WAK1 gene expression in roots. Short- and long-term analysis of gene expression in root fractions showed a typical “on” and “off” pattern with a first peak at 3 h of Al exposure followed by a sharp decline at 6 h and a complete disappearance after 9 h of Al exposure, suggesting the WAK1 is a further representative of Al-induced early genes. In shoots, upon root Al exposure, an increased but stable WAK1 expression was observed. Using confocal microscopy, we visualized Al-induced closure of leaf stomata, consistent with previous suggestions that the Al stress primarily experienced in roots associated with the transfer of root-shoot signals. Elevated levels of WAK protein in root cells were observed through western blots after 6 h of Al exposure, indicating a lag time between the Al-induced WAK transcription and translation. WAK proteins are localized abundantly to peripheries of cortex cells within the elongation zone of the root apex. In these root cells, disintegration of cortical microtubules was observed after Al treatment but not after the Al analog lanthanum treatments. Tip-growing control root hairs, stem stomata, and leaf stomatal pores are characterized with high amounts of WAKs, suggesting WAKs are accumulating at plasma membrane domains, which suffer from mechanical stress and lack dense arrays of supporting cortical microtubules. Further, transgenic plants overexpressing WAK1 showed an enhanced Al tolerance in terms of root growth when compared with the wild-type plants, making the WAK1 one of the important candidates for plant defense against Al toxicity.

Root apex transition zone: a signalling-response nexus in the root.

Longitudinal zonation, as well as a simple and regular anatomy, are hallmarks of the root apex. Here we focus on one particular root-apex zone, the transition zone, which is located between the apical meristem and basal elongation region. This zone has a unique role as the determiner of cell fate and root growth; this is accomplished by means of the complex system of a polar auxin transport circuit. The transition zone also integrates diverse inputs from endogenous (hormonal) and exogenous (sensorial) stimuli and translates them into signalling and motoric outputs as adaptive differential growth responses. These underlie the root-apex tropisms and other aspects of adaptive root behaviour.

Endocytosis and vesicle trafficking during tip growth of root hairs

Summary.The directional elongation of root hairs, “tip growth”, depends on the coordinated and highly regulated trafficking of vesicles which fill the tip cytoplasm and are active in secretion of cell wall material. So far, little is known about the dynamics of endocytosis in living root hairs. We analyzed the motile behaviour of vesicles in the apical region of living root hairs of Arabidopsis thaliana and of Triticum aestivum by live cell microscopy. For direct observation of endocytosis and of the fate of endocytic vesicles, we used the fluorescent endocytosis marker dyes FM 1-43 and FM 4-64. Rapid endocytosis was detected mainly in the tip, where it caused a bright fluorescence of the apical cytoplasm. The internalized membranes proceeded through highly dynamic putative early endosomes in the clear zone to larger endosomal compartments in the subapical region that are excluded from the clear zone. The internalized cargo ended up in the dynamic vacuole by fusion of large endosomal compartments with the tonoplast. Before export to these lytic compartments, putative early endosomes remained in the apical zone, where they most probably recycled to the plasma membrane and back into the cytoplasm for more than 30 min. Endoplasmic reticulum was not involved in trafficking pathways of endosomes. Actin cytoskeleton was needed for the endocytosis itself, as well as for further membrane trafficking. The actin-depolymerizing drug latrunculin B modified the dynamic properties of vesicles and endosomes; they became immobilized and aggregated in the tip. Treatment with brefeldin A inhibited membrane trafficking and caused the disappearance of FM-containing vesicles and putative early endosomes from the clear zone; labelled structures accumulated in motile brefeldin A-induced compartments. These large endocytic compartments redispersed upon removal of the drug. Our results hence prove that endocytosis occurs in growing root hairs. We show the localization of endocytosis in the tip and indicate specific endomembrane compartments and their recycling.

Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth

Mitogen‐activated protein kinases (MAPKs) are involved in stress signaling to the actin cytoskeleton in yeast and animals. We have analyzed the function of the stress‐activated alfalfa MAP kinase SIMK in root hairs. In epidermal cells, SIMK is predominantly nuclear. During root hair formation, SIMK was activated and redistributed from the nucleus into growing tips of root hairs possessing dense F‐actin meshworks. Actin depolymerization by latrunculin B resulted in SIMK relocation to the nucleus. Conversely, upon actin stabilization with jasplakinolide, SIMK co‐localized with thick actin cables in the cytoplasm. Importantly, latrunculin B and jasplakinolide were both found to activate SIMK in a root‐derived cell culture. Loss of tip‐focused SIMK and actin was induced by the MAPK kinase inhibitor UO 126 and resulted in aberrant root hairs. UO 126 inhibited targeted vesicle trafficking and polarized growth of root hairs. In contrast, overexpression of gain‐of‐function SIMK induced rapid tip growth of root hairs and could bypass growth inhibition by UO 126. These data indicate that SIMK plays a crucial role in root hair tip growth.

Root cytoskeleton: its role in perception of and response to gravity

Abstract. We have critically evaluated the possible functions of the plant cytoskeleton in root gravisensing and graviresponse and discussed the evidence that microtubules (MTs) and actin microfilaments (MFs) do not control differential cell growth during bending of roots. On the other hand, MF and MT networks are envisaged to participate in gravisensing because of the mechanical properties of the cytoskeletal structures that interconnect plant cell organelles with the plasma membrane. In restrained gravisensing, forces are suggested to be transmitted to membranes because large-scale gravity-dependent repositioning of organelles is effectively prevented due to the cytoskeleton-mediated anchorage of their envelopes at the plasma membrane. From the cytoskeletal point of view, we can also envisage an unrestrained gravity sensing when cytoskeletal tethers are not strong enough to preserve the tight control over distribution of organelles and the latter, if heavy enough, are allowed to sediment towards the physical bottom of cells. This situation obviously occurs in root cap statocytes because these uniquely organized cells are depleted of prominent actin MF bundles, endoplasmic MT arrays, and ER elements in their internal cytoplasm. Nevertheless, indirect evidence clearly indicates that sedimented root cap statoliths are enmeshed within fine but dynamic MF networks and that their behaviour is obviously under, at least partial, cytoskeletal control. The actomyosin-enriched domain among and around amyloplasts is proposed to increase the perception of gravity due to the grouping effect of sedimenting statoliths. Cytoskeletal links between myosin-rich statoliths, and cell peripheries well equipped with dense cortical MTs, membrane-associated cytoskeleton, as well as with ER elements, would allow efficient restrained gravisensing only at the statocyte cell cortex. As a consequence of cytoskeletal depletion in the internal statocyte cytoplasm and bulk sedimentation of large amyloplasts, restrained gravisensing is spatially restricted to the bottom of the statocyte irrespective of whether roots are vertical or horizontal. This spatial aspect allows for efficient gravisensing via amplification of gravity-induced impacts on the cellular architecture, a phenomenon which is unique to root cap statocytes.

The Root Apex of Arabidopsis thaliana Consists of Four Distinct Zones of Growth Activities: Meristematic Zone, Transition Zone, Fast Elongation Zone and Growth Terminating Zone.

In the growing apex of Arabidopsis thaliana primary roots, cells proceed through four distinct phases of cellular activities. These zones and their boundaries can be well defined based on their characteristic cellular activities. The meristematic zone comprises, and is limited to, all cells that undergo mitotic divisions. Detailed in vivo analysis of transgenic lines reveals that, in the Columbia-0 ecotype, the meristem stretches up to 200 µm away from the junction between root and root cap (RCJ). In the transition zone, 200 to about 520 µm away from the RCJ, cells undergo physiological changes as they prepare for their fast elongation. Upon entering the transition zone, they progressively develop a central vacuole, polarize the cytoskeleton and remodel their cell walls. Cells grow slowly during this transition: it takes ten hours to triplicate cell length from 8.5 to about 35 µm in the trichoblast cell files. In the fast elongation zone, which covers the zone from 520 to about 850 µm from the RCJ, cell length quadruplicates to about 140 µm in only two hours. This is accompanied by drastic and specific cell wall alterations. Finally, root hairs fully develop in the growth terminating zone, where root cells undergo a minor elongation to reach their mature lengths.