Dieter Volkmann

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.

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.

Actin Cytoskeleton in Plants: From Transport Networks to Signaling Networks

The plant actin cytoskeleton is characterized by a high diversity in regard to gene families, isoforms, and degree of polymerization. In addition to the most abundant F‐actin assemblies like filaments and their bundles, G‐actin obviously assembles in the form of actin oligomers composed of a few actin molecules which can be extensively cross‐linked into complex dynamic meshworks. The role of the actomyosin complex as a force generating system — based on principles operating as in muscle cells — is clearly established for long‐range mass transport in large algal cells and specialized cell types of higher plants. Extended F‐actin networks, mainly composed of F‐actin bundles, are the structural basis for this cytoplasmic streaming of high velocities On the other hand, evidence is accumulating that delicate meshworks built of short F‐actin oligomers are critical for events occurring at the plasma membrane, e.g., actin interventions into activities of ion channels and hormone carriers, signaling pathways based on phospholipids, and exo‐ and endocytotic processes. These unique F‐actin arrays, constructed by polymerization‐depolymerization processes propelled via synergistic actions of actin‐binding proteins such as profilin and actin depolymerizing factor (ADF)/cofilin are supposed to be engaged in diverse aspects of plant morphogenesis. Finally, rapid rearrangements of F‐actin meshworks interconnecting endocellular membranes turn out to be especially important for perception‐signaling purposes of plant cells, e.g., in association with guard cell movements, mechano‐ and gravity‐sensing, plant host–pathogen interactions, and wound‐healing. Microsc. Res. Tech. 47:135–154, 1999.

Oriented movement of statoliths studied in a reduced gravitational field during parabolic flights of rockets.

During five rocket flights (TEXUS 18, 19, 21, 23 and 25), experiments were performed to investigate the behaviour of statoliths in rhizoids of the green alga Chara globularia Thuill. and in statocytes of cress (Lepidium sativum L.) roots, when the gravitational field changed to approx. 10−4 · g (i.e. microgravity) during the parabolic flight (lasting for 301–390 s) of the rockets. The position of statoliths was only slightly influenced by the conditions during launch, e.g. vibration, acceleration and rotation of the rocket. Within approx. 6 min of microgravity conditions the shape of the statolith complex in the rhizoids changed from a transversely oriented lens into a longitudinally oriented spindle. The center of the statolith complex moved approx. 14 μm and 3.6 μm in rhizoids and root statocytes, respectively, in the opposite direction to the originally acting gravity vector. The kinetics of statolith displacement in rhizoids demonstrate that the velocity was nearly constant under microgravity whereas it decreased remarkably after inversion of rhizoids on Earth. It can be concluded that on Earth the position of statoliths in both rhizoids and root statocytes depends on the balance of two forces, i.e. the gravitational force and the counteracting force mediated by microfilaments.

The Signal Transducer NPH3 Integrates the Phototropin1 Photosensor with PIN2-Based Polar Auxin Transport in Arabidopsis Root Phototropism

This work examines blue light–induced root phototropism, finding that it requires the phot1/NONPHOTOTROPIC HYPOCOTYL3 signaling pathway, which stimulates shootward auxin flux by changing PIN2 subcellular localization in the root apex transition zone. Under blue light (BL) illumination, Arabidopsis thaliana roots grow away from the light source, showing a negative phototropic response. However, the mechanism of root phototropism is still unclear. Using a noninvasive microelectrode system, we showed that the BL sensor phototropin1 (phot1), the signal transducer NONPHOTOTROPIC HYPOCOTYL3 (NPH3), and the auxin efflux transporter PIN2 were essential for BL-induced auxin flux in the root apex transition zone. We also found that PIN2-green fluorescent protein (GFP) localized to vacuole-like compartments (VLCs) in dark-grown root epidermal and cortical cells, and phot1/NPH3 mediated a BL-initiated pathway that caused PIN2 redistribution to the plasma membrane. When dark-grown roots were exposed to brefeldin A (BFA), PIN2-GFP remained in VLCs in darkness, and BL caused PIN2-GFP disappearance from VLCs and induced PIN2-GFP-FM4-64 colocalization within enlarged compartments. In the nph3 mutant, both dark and BL BFA treatments caused the disappearance of PIN2-GFP from VLCs. However, in the phot1 mutant, PIN2-GFP remained within VLCs under both dark and BL BFA treatments, suggesting that phot1 and NPH3 play different roles in PIN2 localization. In conclusion, BL-induced root phototropism is based on the phot1/NPH3 signaling pathway, which stimulates the shootward auxin flux by modifying the subcellular targeting of PIN2 in the root apex transition zone.