Arabidopsis thaliana is a widely used model plant for plant biology research. Under traditional agar-plate culture system (TPG, traditional plant-growing), both plant shoots and roots are exposed to illumination, and roots are grown in sucrose-added medium. This is not a natural environment for the roots and may cause artifact responses. We have developed an improved agar-plate culture system (IPG, improved plant-growing) where shoots are illuminated but roots are grown in darkness without sucrose addition. Compared to TPG, IPG produced plants with significantly less total root length, lateral root length and root hair density, although their primary roots were longer. Root gravitropism, PIN2 (an auxin efflux carrier) abundance, H+ efflux or Ca2+ influx in root apexes, were weaker in IPG-grown roots than those in TPG-grown roots. We conclude that IPG offers a more natural way to study the root growth and response of Arabidopsis thaliana.
Primary roots of young maize seedlings showed peculiar growth behavior when challenged by placing them on a slope, or if whole seedlings were turned upside down. Importantly, this behavior was dependent on the light conditions. If roots were placed on slopes in the dark, they performed “crawling” behavior and advanced rapidly up the slope. However, as soon as these roots were illuminated, their crawling movements along their horizontal paths slowed down, and instead tried to grow downwards along the gravity vector. A similar light-induced switch in the root behavior was observed when roots were inverted, by placing them in thin glass capillaries. As long as they were kept in the darkness, they showed rapid growth against the gravity vector. If illuminated, these inverted roots rapidly accomplished U-turns and grew down along the gravity vector, eventually escaping from the capillaries upon reaching their open ends. De-capped roots, although growing vigorously, did not display these light-induced photophobic growth responses. We can conclude that intact root cap is essential for the photophobic root behavior in maize.
gravity; light; phototropism; plant behavior; roots; signaling
Soil alkalinity is a widespread environmental problem that limits agricultural productivity. The hypothesis that an auxin-regulated proton secretion by plasma membrane H+-ATPase plays an important role in root adaption to alkaline stress was studied. It was found that alkaline stress increased auxin transport and PIN2 (an auxin efflux transporter) abundance in the root tip of wild-type Arabidopsis plants (WT). Compared with WT roots, the pin2 mutant roots exhibited much reduced plasma membrane H+-ATPase activity, root elongation, auxin transport, and proton secretion under alkaline stress. More importantly, roots of the pks5 mutant (PKS5, a protein kinase) lacking PIN2 (a pks5/pin2 double mutant) lost the previous higher proton-secretion capacity and higher elongation rate of primary roots under alkaline stress. By using Arabidopsis natural accessions with a high proton-secretion capacity, it was found that their PIN2 transcription abundance is positively related to the elongation rate of the primary root and proton-secretion capacity under alkaline stress. Taken together, our results confirm that PIN2 is involved in the PKS5-mediated signalling cascade under alkaline-stress and suggest that PIN2 is required for the adaptation of roots to alkaline stress by modulating proton secretion in the root tip to maintain primary root elongation.
Alkaline stress; auxin transport; PIN2; PKS5; plasma membrane H+-ATPase; primary root growth; proton secretion; root tip
During seed imbibition and embryo activation, rapid change from a metabolically resting state to the activation of diverse extracellular and/or membrane bound molecules is essential and, hence, endocytosis could be activated too. In fact, we have documented endocytic internalization of the membrane impermeable endocytic tracer FM4–64 already upon 30 min of imbibition of Arabidopsis seeds. This finding suggest that endocytosis is activated early during seed imbibition in Arabidopsis. Immunolocalization of rhamnogalacturonan-II (RG-II) complexed with boron showed that whereas this pectin is localized only in the cell walls of dry seed embryos, it starts to be intracellular once the imbibition started. Brefeldin A (BFA) exposure resulted in recruitment of the intracellular RG-II pectin complexes into the endocytic BFA-induced compartments, confirming the endocytic origin of the RG-II signal detected intracellularly. Finally, germination was significantly delayed when Arabidopsis seeds were germinated in the presence of inhibitors of endocytic pathways, suggesting that trafficking of extracellular molecules might play an important role in the overcome of germination. This work constitutes the first demonstration of endocytic processes during germination and opens new perspectives about the role of the extracellular matrix and membrane components in seed germination.
brefeldin A; endocytosis; extracellular matrix; germination; rhamnogalacturonan-II
To investigate the distribution of IAA (indole-3-acetic acid) and the IAA synthetic cells in maize coleoptiles, we established immunohistochemistry of IAA using an anti-IAA-C-monoclonal antibody. We first confirmed the specificity of the antibody by comparing the amounts of endogenous free and conjugated IAA to the IAA signal obtained from the IAA antibody. Depletion of endogenous IAA showed a corresponding decrease in immuno-signal intensity and negligible cross-reactivity against IAA-related compounds, including tryptophan, indole-3-acetamide, and conjugated-IAA was observed. Immunolocalization showed that the IAA signal was intense in the approximately 1 mm region and the outer epidermis at the approximately 0.5 mm region from the top of coleoptiles treated with 1-N-naphthylphthalamic acid. By contrast, the IAA immuno-signal in the outer epidermis almost disappeared after 5-methyl-tryptophan treatment. Immunogold labeling of IAA with an anti-IAA-N-polyclonal antibody in the outer-epidermal cells showed cytoplasmic localization of free-IAA, but none in cell walls or vacuoles. These findings indicated that IAA is synthesized in the 0–2.0 mm region of maize coleoptile tips from Trp, in which the outer-epidermal cells of the 0.5 mm tip are the most active IAA synthetic cells.
1-N-naphthylphthalamic acid; 5-methyl-tryptophan; Coleoptiles; IAA biosynthesis; Immunohistochemistry of IAA; Indole-3-acetic acid (IAA); Maize
Arabidopsis roots are routinely exposed to light both during their cultivation within transparent Petri dishes and during their confocal microscopy analysis. Here we report that illumination of roots which naturally grow in darkness, even for a few seconds, induces an immediate and strong burst of reactive oxygen species (ROS). Plant scientists studying roots should pay great attention to the environment of living roots, and keep them in darkness as long as possible. Results obtained using illuminated roots during in vivo microscopic analysis should also be interpreted with great caution.
reactive oxygen species; root; light; superoxide; escape phototropism
Different theories have been put forward during the last decade to explain the functional meaning of sleep and dreaming in humans. In the present paper, a new theory is presented which, while taking advantage of these earlier theories, introduces the following new and original aspects:
• Circadian rhythms relevant to various organs of the body affect the reciprocal interactions which operate to maintain constancy of the internal milieu and thereby also affect the sleep/wakefulness cycle. Particular attention is given to the constancy of natraemia and osmolarity and to the permissive role that the evolution of renal function has had for the evolution of the central nervous system and its integrative actions.
• The resetting of neuro-endocrine controls at the onset of wakefulness leads to the acquisition of new information and its integration within previously stored memories. This point is dealt with in relation to Moore-Ede’s proposal for the existence of a ’predictive homeostasis’.
• The concept of ‘psychic homeostasis’ is introduced and is considered as one of the most important states since it is aimed at the well-being, or eudemonia, of the human psyche. Sleep and dreaming in humans are discussed as important functions for the maintenance of a newly proposed composite state: that of ‘predictive psychic homeostasis’.
On the basis of these assumptions, and in accordance with the available neurobiological data, the present paper puts forward the novel hypothesis that sleep and dreaming play important functions in humans by compensating for psychic allostatic overloads. Hence, both consolatory dreams and disturbing nightmares can be part of the vis medicatrix naturae, the natural healing power, in this case, the state of eudemonia.
sleep and dream theories; predictive psychic homeostasis; evolutionary tinkering; homeostasis of internal milieu; circadian rhythms of peripheral organs
Biological evolution represents one of the most successful, but also controversial scientific concepts. Ever since Charles Darwin formulated his version of evolution via natural selection, biological sciences experienced explosive development and progress. First of all, although Darwin could not explain how traits of organisms, selected via natural selection, are inherited and passed down along generations; his theory stimulated research in this respect and resulted in the establishment of genetics and still later in the discovery of DNA and genome sequencing some hundred years after his evolutionary theory. Nevertheless, there are several weaknesses in classical Darwinian as well as Neodarwinian gene-centric views of biological evolution. The most serious drawback is its narrow focus: the modern evolutionary synthesis, as formulated in the 20th Century, is based on the concept of gene and on the mathematical/statistical analysis of populations. While Neodarwinism is still generally considered a valid theory of biological evolution, its narrow focus and incompatibility with several new findings and discoveries calls for its update and/or transformation. Either it will be replaced with an updated version or, if not flexible enough, it will be replaced by a new theory. In his book “Evolution — A New View from the 21st Century,”1 James A. Shapiro discusses these problems as well as newly emerging results which are changing our understanding of biological evolution. This new book joins a row of several other recent books highlighting the same issues.2–13
Monoterpenes at high atmospheric concentrations are strong growth inhibitors in allelopathic interactions. Effects depend on dose, molecular structure of the monoterpene and on the species of the receiver plant. Stomata are among the first targets affected by camphor and menthol. Previously, it could be demonstrated that the compounds induce swelling of the protoplasts, prevent stomatal closure and enhance transpiration. In this study, we show that the block of stomatal closure is accompanied by changes to the cytoskeleton, which has a direct role in stomatal movements. Although MPK3 (MAP3 kinase) and ABF4 gene expressions are induced within six hours, stomatal closure is prevented. In contrast to ABF4, ABF2 (both transcription factors) is not induced. MPK3 and ABF4 both encode for proteins involved in the process of stomatal closure. The expression of PEPCase, an enzyme important for stomatal opening, is downregulated. The leaves develop stress symptoms, mirrored by transient changes in the expression profile of additional genes: lipoxygenase 2 (LOX2), CER5, CER6 (both important for wax production) and RD29B (an ABA inducible stress protein). Non-invasive methods showed a fast response of the plant to camphor fumigations both in a rapid decrease of the quantum yield and in the relative growth rate. Repeated exposures to the monoterpenes resulted finally in growth reduction and a stress related change in the phenotype. It is proposed that high concentrations or repeated exposure to monoterpenes led to irreversible damages, whereas low concentrations or short-term fumigations may have the potential to strengthen the plant fitness.
monoterpene; cytoskeleton; gene expression; phenotype modulation
Plant cells and neurons share several similarities, including non-centrosomal microtubules, motile post-Golgi organelles, separated both spatially/structurally and functionally from the Golgi apparatus and involved in vesicular endocytic recycling, as well as cell-cell adhesion domains based on the actin/myosin cytoskeleton which serve for cell-cell communication. Tip-growing plant cells such as root hairs and pollen tubes also resemble neurons extending their axons. Recently, surprising discoveries have been made with respect to the molecular basis of neurodegenerative disorders known as Hereditary Spastic Paraplegias and tip-growth of root hairs. All these advances are briefly discussed in the context of other similarities between plant cells and neurons.
plant cells; neurons; polarity; tip-growth; Golgi apparatus; endoplasmic reticulum
This year celebrates the 200th aniversary of the birth of Charles Darwin, best known for his theory of evolution summarized in On the Origin of Species. Less well known is that, in the second half of his life, Darwin’s major scientific focus turned towards plants. He wrote several books on plants, the next-to-last of which, The Power of Movement of Plants, published together with his son Francis, opened plants to a new view. Here we amplify the final sentence of this book in which the Darwins proposed that: “It is hardly an exaggeration to say that the tip of the radicle thus endowed [with sensitivity] and having the power of directing the movements of the adjoining parts, acts like the brain of one of the lower animals; the brain being seated within the anterior end of the body, receiving impressions from the sense-organs, and directing the several movements.” This sentence conveys two important messages: first, that the root apex may be considered to be a ‘brain-like’ organ endowed with a sensitivity which controls its navigation through soil; second, that the root apex represents the anterior end of the plant body. In this article, we discuss both these statements.
auxin; cognition; plant neurobiology; plant tropisms; roots; sensory biology; signaling
Mitochondria are dynamic organelles that move along actin filaments, and serve as calcium stores in plant cells. The positioning and dynamics of mitochondria depend on membrane-cytoskeleton interactions, but it is not clear whether microfilament cytoskeleton has a direct effect on mitochondrial function and Ca2+ storage. Therefore, we designed a series of experiments to clarify the effects of actin filaments on mitochondrial Ca2+ storage, cytoplasmic Ca2+ concentration ([Ca2+]c), and the interaction between mitochondrial Ca2+ and cytoplasmic Ca2+ in Arabidopsis root hairs.
In this study, we found that treatments with latrunculin B (Lat-B) and jasplakinolide (Jas), which depolymerize and polymerize actin filaments respectively, decreased membrane potential and Ca2+ stores in the mitochondria of Arabidopsis root hairs. Simultaneously, these treatments induced an instantaneous increase of cytoplasmic Ca2+, followed by a continuous decrease. All of these effects were inhibited by pretreatment with cyclosporin A (Cs A), a representative blocker of the mitochondrial permeability transition pore (mPTP). Moreover, we found there was a Ca2+ concentration gradient in mitochondria from the tip to the base of the root hair, and this gradient could be disrupted by actin-acting drugs.
Based on these results, we concluded that the disruption of actin filaments caused by Lat-B or Jas promoted irreversible opening of the mPTP, resulting in mitochondrial Ca2+ release into the cytoplasm, and consequent changes in [Ca2+]c. We suggest that normal polymerization and depolymerization of actin filaments are essential for mitochondrial Ca2+ storage in root hairs.
plant neurobiology; signaling; behavior
From a structural standpoint, living organisms are organized like a nest of Russian matryoshka dolls, in which structures are buried within one another. From a temporal point of view, this type of organization is the result of a history comprised of a set of time backcloths which have accompanied the passage of living matter from its origins up to the present day. The aim of the present paper is to indicate a possible course of this ‘passage through time, and suggest how today’s complexity has been reached by living organisms. This investigation will employ three conceptual tools, namely the Mosaic, Self-Similarity Logic, and the Biological Attraction principles. Self-Similarity Logic indicates the self-consistency by which elements of a living system interact, irrespective of the spatiotemporal level under consideration. The term Mosaic indicates how, from the same set of elements assembled according to different patterns, it is possible to arrive at completely different constructions: hence, each system becomes endowed with different emergent properties. The Biological Attraction principle states that there is an inherent drive for association and merging of compatible elements at all levels of biological complexity. By analogy with the gravitation law in physics, biological attraction is based on the evidence that each living organism creates an attractive field around itself. This field acts as a sphere of influence that actively attracts similar fields of other biological systems, thereby modifying salient features of the interacting organisms. Three specific organizational levels of living matter, namely the molecular, cellular, and supracellular levels, have been considered in order to analyse and illustrate the interpretative as well as the predictive roles of each of these three explanatory principles.
Self-Similarity Logic Principle; Mosaic Principle; Biological Complexity; Biological Attraction Principle; Dissipative Systems; Endosymbiosis; Evolution; Self-Assembly; Self-Organization
Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements.
We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria.
Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.
NO is an important regulatory molecule in eukaryotes. Much of its effect is ascribed to the action of NO as a signalling molecule. However, NO can also directly modify proteins thus affecting their activities. Although the signalling functions of NO are relatively well recognized in plants, very little is known about its potential influence on the structural integrity of plant cells. In this study, the reorganization of the actin cytoskeleton, and the recycling of wall polysaccharides in plants via the endocytic pathway in the presence of NO or NO-modulating substances were analysed. The actin cytoskeleton and endocytosis in maize (Zea mays) root apices were visualized with fluorescence immunocytochemistry. The organization of the actin cytoskeleton is modulated via NO levels and the extent of such modulation is cell-type specific. In endodermis cells, actin cables change their orientation from longitudinal to oblique and cellular cross-wall domains become actin-depleted/depolymerized. The reaction is reversible and depends on the type of NO donor. Actin-dependent vesicle trafficking is also affected. This was demonstrated through the analysis of recycled wall material transported to newly-formed cell plates and BFA compartments. Therefore, it is concluded that, in plant cells, NO affects the functioning of the actin cytoskeleton and actin-dependent processes. Mechanisms for the reorganization of the actin cytoskeleton are cell-type specific, and such rearrangements might selectively impinge on the functioning of various cellular domains. Thus, the dynamic actin cytoskeleton could be considered as a downstream effector of NO signalling in cells of root apices.
Actin; cell wall–cytoskeleton interactions; endocytosis; maize; nitric oxide; Zea mays
It is generally assumed, both in common-sense argumentations and scientific concepts, that brains and neurons represent late evolutionary achievements which are present only in more advanced animals. Here we overview recently published data clearly revealing that our understanding of bacteria, unicellular eukaryotic organisms, plants, brains and neurons, rooted in the Aristotelian philosophy is flawed. Neural aspects of biological systems are obvious already in bacteria and unicellular biological units such as sexual gametes and diverse unicellular eukaryotic organisms. Altogether, processes and activities thought to represent evolutionary ‘recent’ specializations of the nervous system emerge rather to represent ancient and fundamental cell survival processes.
bacteria; evolution; neuron; neurosciences; plants
The plant hormone auxin is secreted in root apices via phospholipase Dζ2 (PLDζ2) activity which produces specific population of phosphatidic acid that stimulates secretion of vesicles enriched with auxin. These vesicles were reported to be localized at plant synapses which are active in auxin secretion, especially at the transition zone of the root apex. There are several implications of this vesicular secretion of auxin. In root apices, auxin emerges as plant neurotransmitter-like signal molecule which coordinates activities of adjacent cells via electric and chemical signaling. Putative quantal release of auxin after electrical stimulation, if confirmed, would be part of neuronal communication between plant cells. As auxin transport across plant synapses is tightly linked with integrated sensory perception of environment, especially of omnipresent gravity and light, this process is proposed to mediate the plant perception of environment. These neuronal features allow sessile plants to integrate multitude of sensory signals into the adaptive behavior of whole plants and the animal-like exploratory behavior of growing roots.
auxin; phospholipase Dζ2; plant development; root apex; secretion; vesicles
Auxin (IAA) is versatile signalling molecule of plants, currently classified as plant hormone. But there are data suggesting that auxin is acting also as plant-specific morphogen, electric-responses inducing transmitter, and as general signalling molecule used for plant-bacteria communication. Our previous data revealed that auxin is associated with secretory endosomes and also highly enriched within cell walls of cells active in transcellular auxin transport. Our present data, based on in vivo non-invasive auxin flux recordings, reveal that auxin is secreted out of synaptic-like domains specialized for efflux of auxin in root apex cells highly active in polar cell-cell transport of auxin. We obtained both genetic and pharmacological evidence that phospholipase Dζ2 drives vesicular secretion of auxin for its polar transcellular transport in the transition zone of the root apex. Secretion of auxin via secretory vesicles has far-reaching consequences not only for our understanding of cell-cell auxin transport but also for plant sciences as a whole.
auxin; Brefeldin A; plant synapse; phospholipase D; phosphatidic acid; secretion; vesicle recycling
Plants are complex living beings, extremely sensitive to environmental factors, continuously adapting to the ever changing environment. Emerging research document that plants sense, memorize, and process experiences and use this information for their adaptive behavior and evolution. As any other living and evolving systems, plants act as knowledge accumulating systems. Neuronal informational systems are behind this concept of organisms as knowledge accumulating systems because they allow the most rapid and efficient adaptive responses to changes in environment. Therefore, it should not be surprising that neuronal computation is not limited to animal brains but is used also by bacteria and plants. The journal, Plant Signaling & Behavior, was launched as a platform for exchanging information and fostering research on plant neurobiology in order to allow our understanding of plants in their whole integrated, communicative, and behavioral complexity.
I always go by official statistics because they are very carefully compounded and, even if they are false, we have no others …∼ Jaroslav Hašek, 1911
plant neurobiology; sensory biology; behavior; biological complexity; evolution; signal integration
• Background and Aims Neutral red (NR), a lipophilic phenazine dye, has been widely used in various biological systems as a vital stain for bright-field microscopy. In its unprotonated form it penetrates the plasma membrane and tonoplast of viable plant cells, then due to protonation it becomes trapped in acidic compartments. The possible applications of NR for confocal laser scanning microscopy (CLSM) studies were examined in various aspects of plant root biology.
• Methods NR was used as a fluorochrome for living roots of Phaseolus vulgaris, Allium cepa, A. porrum and Arabidopsis thaliana (wild-type and transgenic GFP-carrying lines). The tissues were visualized using CLSM. The effect of NR on the integrity of the cytoskeleton and the growth rate of arabidopsis primary roots was analysed to judge potential toxic effects of the dye.
• Key Results The main advantages of the use of NR are related to the fact that NR rapidly penetrates root tissues, has affinity to suberin and lignin, and accumulates in the vacuoles. It is shown that NR is a suitable probe for visualization of proto- and metaxylem elements, Casparian bands in the endodermis, and vacuoles in cells of living roots. The actin cytoskeleton and the microtubule system of the cells, as well as the dynamics of root growth, remain unchanged after short-term application of NR, indicating a relatively low toxicity of this chemical. It was also found that NR is a useful probe for the observation of the internal structures of root nodules and of fungal hyphae in vesicular–arbuscular mycorrhizas.
• Conclusions Ease, low cost and absence of tissue processing make NR a useful probe for structural, developmental and vacuole-biogenetic studies of plant roots with CLSM.
CLSM; Casparian band; xylem; vacuole biogenesis; lateral root development; root development; neutral red
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.
Arabidopsis; cytoskeleton; development; differentiation zone; elongation zone; growth; growth terminating zone; meristem; root apex; transition zone
Glycogen synthase kinase 3 (GSK-3) was originally identified as a regulator of glycogen synthesis in mammals. Like starch in plants, glycogen is a polymer of glucose, and serves as an energy and carbon store. Starch is the main carbohydrate store in plants. Regulation of starch metabolism, in particular in response to environmental cues, is of primary importance for carbon and energy flow in plants but is still obscure. Here, we provide evidence that MsK4, a novel Medicago sativa GSK-3-like kinase, connects stress signalling with carbon metabolism. MsK4 was found to be a plastid-localized protein kinase that is associated with starch granules. High-salt stress rapidly induced the in vivo kinase activity of MsK4. Metabolic profiling of MsK4 over-expressor lines revealed changes in sugar metabolism, including increased amounts of maltose, the main degradation product of starch in leaves. Plants over-expressing MsK4 showed improved tolerance to salt stress. Moreover, under high-salinity conditions, MsK4-over-expressing plants accumulated significantly more starch and showed modified carbohydrate content compared with wild-type plants. Overall, these data indicate that MsK4 is an important regulator that adjusts carbohydrate metabolism to environmental stress.
signal transduction; protein phosphorylation; starch