Plant Science View on Biohybrid Development.

Biohybrid consists of a living organism or cell and at least one engineered component. Designing robot-plant biohybrids is a great challenge: it requires interdisciplinary reconsideration of capabilities intimate specific to the biology of plants. Envisioned advances should improve agricultural/horticultural/social practice and could open new directions in utilization of plants by humans. Proper biohybrid cooperation depends upon effective communication. During evolution, plants developed many ways to communicate with each other, with animals, and with microorganisms. The most notable examples are: the use of phytohormones, rapid long-distance signaling, gravity, and light perception. These processes can now be intentionally re-shaped to establish plant-robot communication. In this article, we focus on plants physiological and molecular processes that could be used in bio-hybrids. We show phototropism and biomechanics as promising ways of effective communication, resulting in an alteration in plant architecture, and discuss the specifics of plants anatomy, physiology and development with regards to the bio-hybrids. Moreover, we discuss ways how robots could influence plants growth and development and present aims, ideas, and realized projects of plant-robot biohybrids.

Journey from the Center of the Cell - the intra- and intercellular transport of mRNA.

Transport and localized translation of mRNA is crucial for the proper spatiotemporal organization of proteins within cells. Distribution of RNAs to subcellular domains has recently emerged as a major mechanism for establishing functionally distinct compartments and structures in the cells. There is an emerging evidence that active transport of mRNA involves cytoskeleton and membrane trafficking pathways in fungi, plants and animals, suggesting that it is a common phenomenon among eukaryotes. The important highlights are that the RNA-binding proteins recognize the cargo mRNA and that RNPs are actively transported on the cytoskeletal tracks or co-transported with membranous compartments, such as the endoplasmic reticulum and endosomes. The interest of scientists has expanded over the past years in response to the discoveries that RNA can be exported from cells to play a role in the intercellular communication. In this review, we will focus on characterization of the RNA transport both, within a cell and between cells, and on the currently proposed mechanisms for RNA targeting.

Nitration of plant apoplastic proteins from cell suspension cultures.

Nitric oxide causes numerous protein modifications including nitration of tyrosine residues. This modification, though one of the greatest biological importance, is poorly recognized in plants and is usually associated with stress conditions. In this study we analyzed nitrotyrosines from suspension cultures of Arabidopsis thaliana and Nicotiana tabacum, treated with NO modulators and exposed to osmotic stress, as well as of BY2 cells long-term adapted to osmotic stress conditions. Using confocal microscopy, we showed that the cell wall area is one of the compartments most enriched in nitrotyrosines within a plant cell. Subsequently, we analyzed nitration of ionically-bound cell-wall proteins and identified selected proteins with MALDI-TOF spectrometry. Proteomic analysis indicated that there was no significant increase in the amount of nitrated proteins under the influence of NO modulators, among them 3-morpholinosydnonimine (SIN-1), considered a donor of nitrating agent, peroxynitrite. Moreover, osmotic stress conditions did not increase the level of nitration in cell wall proteins isolated from suspension cells, and in cultures long-term adapted to stress conditions; that level was even reduced in comparison with control samples. Among identified nitrotyrosine-containing proteins dominated the ones associated with carbon circulation as well as the numerous proteins responding to stress conditions, mainly peroxidases. BIOLOGICAL SIGNIFICANCE: High concentrations of nitric oxide found in the cell wall and the ability to produce large amounts of ROS make the apoplast a site highly enriched in nitrotyrosines, as presented in this paper. Analysis of ionically bound fraction of the cell wall proteins indicating generally unchanged amounts of nitrotyrosines under influence of NO modulators and osmotic stress, is noticeably different from literature data concerning, however, the total plant proteins analysis. This observation is supplemented by further nitroproteome analysis, for cells long-term adapted to stressful conditions, and results showing that such conditions did not always cause an increase in nitrotyrosine content. These findings may be interpreted as characteristic features of apoplastic protein nitration.

Collagenase as a useful tool for the analysis of plant cellular peripheries.

A technique for the selective loosening of the cell wall structure and the isolation of proteins permanently knotted in the cell walls was elaborated. Following treatment with collagenase, some proteins, such as calreticulin (CRT) and auxin binding protein 1 (ABP1) were released from purified cell walls, most probably through destruction of respective interacting proteins. The results were confirmed by the immunolocalization of the ABP1 and CRT with confocal and electron microscopy. On the other hand, potential substrates of collagenase, among them annexin 1 have been recognized. Mass spectra of annexin 1 obtained after collagenase digestion and results from analysis of potential cleavage sites suggested that the mechanism of enzyme cleavage might not depend on the amino acid sequence. Summarizing, collagenase was found to be a very useful tool for exploring molecules involved in the functioning of cellular peripheries.

Gamma-secretase subunits associate in intracellular membrane compartments in Arabidopsis thaliana.

Gamma-secretase is a multisubunit complex with intramembrane proteolytic activity. In humans it was identified in genetic screens of patients suffering from familial forms of Alzheimer's disease, and since then it was shown to mediate cleavage of more than 80 substrates, including amyloid precursor protein or Notch receptor. Moreover, in animals, γ-secretase was shown to be involved in regulation of a wide range of cellular events, including cell signalling, regulation of endocytosis of membrane proteins, their trafficking, and degradation. Here we show that genes coding for γ-secretase homologues are present in plant genomes. Also, amino acid motifs crucial for γ-secretase activity are conserved in plants. Moreover, all γ-secretase subunits: PS1/PS2, APH-1, PEN-2, and NCT colocalize and interact with each other in Arabidopsis thaliana protoplasts. The intracellular localization of γ-secretase subunits in Arabidopsis protoplasts revealed a distribution in endomembrane system compartments that is consistent with data from animal studies. Together, our data may be considered as a starting point for analysis of γ-secretase in plants.

The very many faces of presenilins and the γ-secretase complex.

Presenilin is a central, catalytic component of the γ-secretase complex which conducts intramembrane cleavage of various protein substrates. Although identified and mainly studied through its role in the development of amyloid plaques in Alzheimer disease, γ-secretase has many other important functions. The complex seems to be evolutionary conserved throughout the Metazoa, but recent findings in plants and Dictyostelium discoideum as well as in archeons suggest that its evolution and functions might be much more diversified than previously expected. In this review, a selective survey of the multitude of functions of presenilins and the γ-secretase complex is presented. Following a brief overview of γ-secretase structure, assembly and maturation, three functional aspects are analyzed: (1) the role of γ-secretase in autophagy and phagocytosis; (2) involvement of the complex in signaling related to endocytosis; and (3) control of calcium fluxes by presenilins.

Plant plasma membrane-bound staphylococcal-like DNases as a novel class of eukaryotic nucleases.

BACKGROUND: The activity of degradative nucleases responsible for genomic DNA digestion has been observed in all kingdoms of life. It is believed that the main function of DNA degradation occurring during plant programmed cell death is redistribution of nucleic acid derived products such as nitrogen, phosphorus and nucleotide bases. Plant degradative nucleases that have been studied so far belong mainly to the S1-type family and were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. However, the explanation of how degraded DNA components are exported from the dying cells for further reutilization remains open. RESULTS: Bioinformatic and experimental data presented in this paper indicate that two Arabidopsis staphylococcal-like nucleases, named CAN1 and CAN2, are anchored to the cell membrane via N-terminal myristoylation and palmitoylation modifications. Both proteins possess a unique hybrid structure in their catalytic domain consisting of staphylococcal nuclease-like and tRNA synthetase anticodon binding-like motifs. They are neutral, Ca2+-dependent nucleaces showing a different specificity toward the ssDNA, dsDNA and RNA substrates. A study of microarray experiments and endogenous nuclease activity revealed that expression of CAN1 gene correlates with different forms of programmed cell death, while the CAN2 gene is constitutively expressed. CONCLUSIONS: In this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. The identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for DNA and RNA degradation. The potential functions of these nucleases in relation to their unique intracellular location are discussed.

[Structural modifications of proteins induced by nitric oxide]. / Modyfikacje strukturalne bialek wywolane przez tlenek azotu.

It is well known for ca. two decades that nitric oxide regulates many life processes both in animals and in plants. The list of processes controlled by NO is steadily expanding, and some of the mechanisms of action of this small molecule are being unravelled and understood. Nitric oxide is exerting its action through addition to the transition metal ions which normally function as protein cofactors; in this way NO regulates, e.g., the activity of cytoplasmic guanyl cyclase. Recently, however, more and more often direct structural modifications of peptidyl amino acid residues are being studied. Particular attention is being paid to the modifications of cysteine (S-nitrosylation) and tyrosine (nitration) residues with respect to their putative signalling functions. It is also known that these modifications are modulating activities of numerous proteins. In this paper we are discussing structural modifications of amino acid residues by NO taking into account the conditions which should be fulfilled to consider their signalling functions. Moreover, we also present available methodologies for their analysis and identifications of modified proteins.

A fluorescence correlation spectroscopy study of ligand interaction with cytokinin-specific binding protein from mung bean.

Cytokinins are essential plant hormones that regulate numerous physiological processes. Recently, a protein was identified in mung bean (Vigna radiata) and characterized as a cytokinin-specific binding protein (VrCSBP). Fluorescence correlation spectroscopy was used to investigate the interaction between VrCSBP and its ligands. The synthetic cytokinin, N-phenyl-N'-(4-pyridyl) urea, was labeled with two fluorophores, 7-nitro-2,1,3-benzoxadiazole and rhodamine B. Protein-ligand binding was analyzed in an equilibrium saturation binding experiment and confirmed by the competition assay. Surprisingly, it was found that VrCSBP binds not only to cytokinins, but also to gibberellins. In addition, in the presence of natural cytokinins and gibberellins, two populations of VrCSBP that differ in their diffusion coefficients were detected. The diffusion coefficients of these two populations could be related to mono- and dimeric states, which suggests a new mode of operation in ligand binding by VrCSBP, in which dimerization induced by natural ligands enhances the ligand binding capacity of the protein.

[Endocytosis and actin-dependent transport of plant cell wall components]. / Endocytoza i zalezny od cytoszkieletu transport skladników scian komórkowych roslin.

Endocytosis is the way of uptaking and transporting substances from the immediate surroundings, as well as the way of removal and/or recycling of the plasma membrane components. For many years, it was thought that this process was not operating in plant cells in which high turgor pressure of the protoplast prevented invagination of the plasma membrane. Recent years, however, brought the evidence for intensive endocytosis in plants. So far, four types of endocytosis, among 5 known, have been identified in plants. Use of protein markers of different compartments of the endomembrane system, specific dyes, and the cytoskeleton inhibitors, made possible demonstration that, as opposed to animal and fungal systems, endocytosis in plant cells was dependent on actin and plant-specific myosins of VIII and XI classes. Although cell divisions in plants and animals differ considerably, they both are dependent on the proper endocytosis. It has been shown that during cytokinesis secretion and endocytosis act in concert, delivering polysaccharides directionally to the growing cell plate. What is more important, new cell walls are built from e.g. pectins transported by endocytosis from existing cell walls.

[At the plant side of formins--organizers of the actin cytoskeleton]. / Roslinna twarz formin--organizatorów cytoszkieletu aktynowego.

Rearrangements of actin cytoskeleton enable proper functioning of the cells under normal conditions, and also cellular adaptations to changes in the direct surroundings. Formins are actin binding proteins, responsible for actin nucleation and further elongation of microfilaments. The distinguishing feature of formins is the presence of conserved FH2 (formin homology domain 2) domain, as well as other domains typical for distinct formin classes. In animal cells formins are involved in cytokinesis and determination and maintenance of the cell shape and polarity, but also in the formation of filopodia, endocytosis and many other processes. The presence of proteins from the formin family in plant cells, and their involvement in the tip growth and cytokinesis, has been determined only recently. As the functional organization of plant and animal cells is different, one can assume that the range of putative functions of plant formins might also be diverse. One of such proposed functions for formins in plants is the role of linker protein within WMC continuum (cell wall-plasma membrane-cytoskeleton). Unfortunately, for that moment the state of knowledge about plant formins in comparison with animal or fungal ones is much poorer.

The Arabidopsis CBP20 targets the cap-binding complex to the nucleus, and is stabilized by CBP80.

The cap-binding protein complex (CBC) binds to the caps of all RNA polymerase II transcripts, and plays an important role in RNA metabolism. We characterized interactions, localization and nuclear-cytoplasmic transport of two subunits of the Arabidopsis thaliana cap-binding protein complex (AtCBC): AtCBP20 and AtCBP80. Using CFP/YFP-tagged proteins, we show that transport of AtCBC from the cytoplasm to the nucleus in the plant cell is different from that described in other eukaryotic cells. We show that the smaller subunit of the complex, AtCBP20, plays a crucial role in the nuclear import of AtCBC. The C-terminal part of AtCBP20 contains two functionally independent nuclear localization signals (NLSs). At least one of these two NLSs is required for the import of CBC into the plant nucleus. The interaction between the A. thaliana CBP20 and CBP80 was also analyzed in detail, using the yeast two-hybrid system and fluorescence resonance energy transfer (FRET) assays. The N-terminal part of AtCBP20 is essential for interaction with AtCBP80. Furthermore, AtCBP80 is important for the protein stability of the smaller subunit of CBC. Based on these data, we propose a model for the nuclear-cytoplasmic trafficking of the CBC complex in plants.

Nitric oxide modulates dynamic actin cytoskeleton and vesicle trafficking in a cell type-specific manner in root apices.

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.

Inhibitors of protein glycosylation or secretion change the pattern of extracellular proteins in suspension-cultured cells of Arabidopsis thaliana.

Cell walls are essential for plant development and morphogenesis. The majority of wall proteins are glycosylated, either as N- or O-glycans. Various inhibitors of glycosylation and secretion are used to determine the importance of wall proteins for the functioning of the walls. Tunicamycin is an inhibitor of the first enzyme in the N-glycosylation pathway, 3,4-dehydroproline inhibits peptidyl proline hydroxylation, and Brefeldin A is an inhibitor of vesicle trafficking, disrupting the delivery of wall polymers to the apoplast. In inhibitor-treated suspension-cultured Arabidopsis thaliana cells, qualitative and quantitative differences in the extracellular proteome were observed for both proteins secreted into medium or ionically-bound in the walls. Lack of O-glycosylation resulted in the selective protein loss from the extracellular compartments. Following tunicamycin treatment the secretion of additional proteins as well as ER-resident chaperones from the Hsp70 and Hsp90 families outside the protoplasts was noted. Moreover, changes in the proteolytic degradation pattern of culture filtrate proteins were also observed. Application of Brefeldin A resulted in transient and selective loss of individual proteins from the extracellular compartments of A. thaliana cell suspension. We conclude that post-translational modifications are vital for the proper functioning of wall proteins. N-glycosylation is crucial for their proper folding and stability. Extracellular compartments could also serve as a sink for improperly folded proteins during the unfolded protein response.

Influence of plant secondary metabolites on in vitro oxidation of methyl ferulate with cell wall peroxidases from lupine apoplast.

Ionically bound cell wall peroxidases (POXs) were liberated to intercellular washing fluids (IWFs) and isolated together with other proteins and metabolites present in the apoplast of white lupine (Lupinus albus L. var. Bac) root. After separation of proteins from low molecular weight compounds, activity of peroxidases was monitored in in vitro experiments. Oxidation of methyl ferulate with H2O2 was studied in multi-component mixtures of plant metabolites. Secondary metabolites identified in IWFs or other natural products playing important roles in different physiological processes were applied as modifiers of the dehydrodimerization process during oxidation reactions performed in vitro. These were isoflavones and their conjugates, lupanine representing quinolizidine alkaloids synthesized in lupine, or other natural products such as quercetin, ascorbic, and salicylic acid. The influence of these substances on the oxidation kinetics of methyl ferulate was monitored with liquid chromatography with ultraviolet detection (LC/UV), and identification of compounds was confirmed with the liquid chromatography/mass spectroscopy (LC/MS) system. On the basis of data collected, it was possible to reveal changes in the activities of cell wall POXs. Application of the LC system permitted us to monitor, independently, quantitative changes of two or more reaction products in the mixtures. In multi-component combinations, oxidation yields of methyl ferulate by POXs were modified depending on the actual composition of the reaction mixture. We conclude that various classes of plant secondary metabolites can modify the yield of methyl ferulate oxidation by hydrogen peroxide in the presence of POX, due to interactions with the enzyme's active site (genistein) or radical scavenging properties of metabolites present in the reaction mixture.

Domain-specific mechanosensory transmission of osmotic and enzymatic cell wall disturbances to the actin cytoskeleton.

Plant protoplasts are embedded within surrounding cell walls and the cell wall-plasma membrane-cytoskeleton (WMC) structural continuum seems to be crucial for the proper functioning of plant cells. We have utilised the protoplast preparation methodology to study the organisation and the putative components of the WMC continuum. Application of an osmotic agent evoked plasmolysis of the Zea mays root apex cells which appeared to be cell type- and growth stage-specific. Simultaneous use of wall polysaccharide-digesting enzymes selectively severed linkages between the components of the WMC continuum which changed the plasmolytic patterns in various cell types. This was followed by a reorganisation of filamentous actin aimed to reinforce protoplast boundaries and maintain the functioning of intercellular contact sites, especially at the cross walls. Particularly strong effects were evoked by pectin-degrading enzymes. Such treatments demonstrated directly the differentiated composition of various wall domains surrounding individual cells with the pectin-enriched cross walls (synapses), and the cellulose-hemicellulose network dominating the side walls. The same wall-degrading enzymes were used for in vitro digestion of isolated Lupinus albus cell walls followed by the extraction of wall proteins. Selective release of proteins suggested the importance of wall polysaccharide-protein interactions in the maintenance of the functioning and mechanical stability of root cell walls.

Recruitment of myosin VIII towards plastid surfaces is root-cap specific and provides the evidence for actomyosin involvement in root osmosensing.

The existence of a cell wall-plasma membrane-cytoskeleton (WMC) continuum in plants has long been postulated. However, the individual molecules building such a continuum are still largely unknown. We test here the hypothesis that the integrin-based multiprotein complexes of animal cells have been replaced in plants with more dynamic entities. Using an experimental approach based on protoplast digestion mixtures, and utilising specific antibodies against Arabidopsis ATM1 myosin, we reveal possible roles played by plant-specific unconventional myosin VIII in the functioning of WMC continuum. We demonstrate rapid relocation (less than 5 min) of myosin VIII to statolith surfaces in maize root-cap cells, which is accompanied by the reorganisation of actin cytoskeleton. Upon prolonged stimulation, myosin VIII is also recruited to plasmodesmata and pit-fields of plasmolysing root cap statocytes. The osmotic stimulus is the major factor inducing relocation, but the cell wall-cytoskeleton interactions also play an important role. In addition, we demonstrate the tight association of myosin VIII with the surfaces of chloroplasts, and provide an indication for the differences in the mechanisms of plastid movement in roots and leaves of plants. Overall, our data provide evidence for the active involvement of actomyosin complexes, rooted in the WMC continuum, in the cellular volume control and maintenance of spatial relationships between cellular compartments.

The architecture of polarized cell growth: the unique status of elongating plant cells.

Polarity is an inherent feature of almost all prokaryotic and eukaryotic cells. In most eukaryotic cells, growth polarity is due to the assembly of actin-based growing domains at particular locations on the cell periphery. A contrasting scenario is that growth polarity results from the establishment of non-growing domains, which are actively maintained at opposite end-poles of the cell. This latter mode of growth is common in rod-shaped bacteria and, surprisingly, also in the majority of plant cells, which elongate along the apical-basal axes of plant organs. The available data indicate that the non-growing end-pole domains of plant cells are sites of intense endocytosis and recycling. These actin-enriched end-poles serve also as signaling platforms, allowing bidirectional exchange of diverse signals along the supracellular domains of longitudinal cell files. It is proposed that these actively remodeled end-poles of elongating plant cells remotely resemble neuronal synapses.