Plant Cell Organelles Research Articles

Immunological evidence for the presence of peroxiredoxin in pea leaf peroxisomes and response to oxidative stress conditions

Peroxiredoxins (Prxs) constitute a group of thiol-specific antioxidant enzymes which are present in bacteria, yeasts, and in plant and animal cells. Although Prxs are mainly localized in the cytosol, they are also present in mitochondria, chloroplasts, and nuclei, but there is no evidence of the existence of Prxs in plant peroxisomes. Using soluble fractions (matrices) of peroxisomes purified from leaves of pea (Pisum sativum L.) plants, the immunological analysis with affinity-purified IgG against yeast Prx1 revealed the presence of an immunoreactive band of about 50 kDa. The apparent molecular mass of the peroxisomal Prx was not sensitive to oxidizing and reducing conditions what could be a mechanism of protection against the oxidative environment existing in peroxisomes. Postembedment, EM immunocytochemical analysis with affinity-purified IgG against yeast Prx1 antibodies, confirmed that this protein was present in the peroxisomal matrix, mitochondria, and chloroplasts. In pea plants grown under oxidative stress conditions, the protein level of peroxisomal Prx was differentially modulated, being slightly induced by growth of plants with 50 µM CdCl2, but being significantly reduced by treatment with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The presence in the matrix of peroxisomes of a protein immunorelated to Prx of about 50 kDa, which is in the range of molecular mass of the dimeric form of other Prxs, opens new questions on the molecular properties of Prxs, but also on their function in the metabolism of reactive oxygen and nitrogen species (ROS/RNS) in these plant cell organelles, where they could be involved in the regulation of hydrogen peroxide and/or peroxynitrite.

Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development.

Plastids display a high morphological and functional diversity. Starting from an undifferentiated small proplastid, these plant cell organelles can develop into four major forms: etioplasts in the dark, chloroplasts in green tissues, chromoplasts in colored flowers and fruits and amyloplasts in roots. The various forms are interconvertible into each other depending on tissue context and respective environmental condition. Research of the last two decades uncovered that each plastid type contains its own specific proteome that can be highly different from that of the other types. Composition of these proteomes largely defines the enzymatic functionality of the respective plastid. The vast majority of plastid proteins is encoded in the nucleus and must be imported from the cytosol. However, a subset of proteins of the photosynthetic and gene expression machineries are encoded on the plastid genome and are transcribed by a complex transcriptional apparatus consisting of phage-type nuclear-encoded RNA polymerases and a bacterial-type plastid-encoded RNA polymerase. Both types recognize specific sets of promoters and transcribe partly over-lapping as well as specific sets of genes. Here we summarize the current knowledge about the sequential activity of these plastid RNA polymerases and their relative activities in different types of plastids. Based on published plastid gene expression profiles we hypothesize that each conversion from one plastid type into another is either accompanied or even preceded by significant changes in plastid transcription suggesting that these changes represent important determinants of plastid morphology and protein composition and, hence, the plastid type.

Vacuolar convolution: possible mechanisms and role of phosphatidylinositol 3,5-bisphosphate.

The central or lytic vacuole is the largest intracellular organelle in plant cells, but we know unacceptably little about the mechanisms regulating its function in vivo. The underlying reasons are related to difficulties in accessing this organelle without disrupting the cellular integrity and to the dynamic morphology of the vacuole, which lacks a defined structure. Among such morphological changes, vacuolar convolution is probably the most commonly observed event, reflected in the (reversible) transformation of a large central vacuole into a structure consisting of interconnected bubbles of a smaller size. Such behaviour is observed in plant cells subjected to hyperosmotic stress but also takes place in physiological conditions (e.g. during stomatal closure). Although vacuolar convolution is a relatively common phenomenon in plants, studies aimed at elucidating its execution mechanisms are rather scarce. In the present review, we analyse the available evidence on the participation of the cellular cytoskeleton and ion transporters in vacuolar morphology dynamics, putting special emphasis on the available evidence of the role played by phosphatidylinositol 3,5-bisphosphate in this process.

Lipid-Protein Microinclusions in the Morphological Structures of Organelle Membranes Studied by Fluorescent Confocal Microscopy

Peculiar properties of morphological structures of organelle membranes were studied by fluorescent confocal microscopy. The list of objects in our experiments was represented by mitochondria, chloroplasts and vacuoles. During this study, identification of lipid microinclusions having the form of such lipid-protein structural microformations as lipid-protein microdomains, vesicles and membrane tubular structures (cytoplasmic transvacuolar strands and nanotubes) located in organelle membranes or bound up with them was conducted. Such membrane probes as laurdan, DPH, ANS and bis-ANS were used. Comparison of fluorescence intensity of these membrane probes was conducted. This investigation of the morphological properties of lipid-protein structural microformations was accompanied with analysis of 1) the phase state and 2) dynamics of microviscosity variations in the membrane elements of isolated plant cell organelles. Distributions of laurdan fluorescence generalized polarization (GP) values for the membrane on the whole and for the intensively fluorescing membrane segments were obtained. It was discovered that the microviscosity of intensively fluorescing membrane segments essentially differed from the microviscosity of the rest part of the membrane. In conclusion, some results of the study of peculiar properties of lipid-protein structural microformations related to the structure of organelle membranes and the discoveries made in this investigation are discussed.

Role of exogenous salicylic acid in regulating physio-morphic and molecular changes under chromium toxicity in black- and yellow- seeded Brassica napus L.

Salicylic acid (SA) mediates tolerance mechanisms in plants against a wide spectrum of biotic and abiotic stresses. Therefore, the present study was carried out to determine how SA regulates the plant protection mechanisms in two cultivars of oilseed rape (Brassica napus L.) under chromium (Cr) stress. Exogenously applied SA enhanced plant growth, increased dry biomasses, and strengthened the reactive oxygen scavenging system by improving cell organelles that were severely damaged via Cr toxicity. The contents of Cr were significantly enhanced in both root and leaf of cultivar Zheda 622 (yellow color) compared with cultivar ZS 758 (black color). Exogenous application of SA significantly reduced the Cr contents in both plant organs as well as enhanced the SA contents under Cr stress. A dose-dependent increase was observed in reactive oxygen species (ROS) generation under Cr stress. To ease the inimical effects of ROS, plants' defense systems were induced under Cr stress, and SA further enhanced protection. Further, TEM micrographs results showed that Cr stress alone significantly ruptured the plant cell organelles of both cultivars by increasing the size of starch grain and the number of plastoglobuli, damaging the chloroplast and mitochondrion structures. However, exogenously applied SA significantly recovered these damages in the plant cells of both cultivars. It was also observed that cultivar ZS 758 was proved to be more tolerant under Cr toxicity. Gene expression analysis revealed that combined treatments of Cr and SA increased antioxidant-related gene expression in both cultivars. Findings of the present study demonstrate that SA induces the enzymatic antioxidant activities and related gene expression, secondary metabolism, and improves the cell structural changes and the transcript level of specific stress-associated proteins in root and leaf of two oilseed rape cultivars under Cr toxicity.

The complete chloroplast genome sequence of Tartary Buckwheat Cultivar Miqiao 1(Fagopyrum tataricum Gaertn.)

Chloroplasts (cp) are indispensable organelles in plant cells that perform photosynthesis amongst other functions, including producing pigments, synthesizing sugars and certain amino acids. The complete chloroplast genome of Tartary Buckwheat Cultivar Miqiao 1 is sequenced in this study. Miqiao 1 is currently the only Tartary Buckwheat variety that can produce the whole nutritive Tartary Buckwheat Rice by hulling. The Miqiao 1 cp genome size is 159,272 bp in length, including 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes. A pair of inverted repeats (IRs) of 30,817 bp were disconnected by a large single copy (LSC) of 84,397 bp and a small single copy (SSC) of 13,241 bp. The cp genome with 37.9% GC content contained 113 annotated genes.

Quantitative Analysis of Microbe-Associated Molecular Pattern (MAMP)-Induced Ca(2+) Transients in Plants.

Ca(2+) is a secondary messenger involved in early signaling events triggered in response to a plethora of biotic and abiotic stimuli. In plants, environmental cues that induce cytosolic Ca(2+) elevation include touch, reactive oxygen species, cold shock, and salt or osmotic stress. Furthermore, Ca(2+) signaling has been implicated in early stages of plant-microbe interactions of both symbiotic and antagonistic nature. A long-standing hypothesis is that there is information encoded in the Ca(2+) signals (so-called Ca(2+) signatures) to enable plants to differentiate between these stimuli and to trigger the appropriate cellular response. Qualitative and quantitative measurements of Ca(2+) signals are therefore needed to dissect the responses of plants to their environment. Luminescence produced by the Ca(2+) probe aequorin upon Ca(2+) binding is a widely used method for the detection of Ca(2+) transients and other changes in Ca(2+) concentrations in cells or organelles of plant cells. In this chapter, using microbe-associated molecular patterns (MAMPs), such as the bacterial-derived flg22 or elf18 peptides as stimuli, a protocol for the quantitative measurements of Ca(2+) fluxes in apoaequorin-expressing seedlings of Arabidopsis thaliana in 96-well format is described.

STEM Tomography Imaging of Hypertrophied Golgi Stacks in Mucilage-Secreting Cells.

Because of the weak penetrating power of electrons, the signal-to-noise ratio of a transmission electron micrograph (TEM) worsens as section thickness increases. This problem is alleviated by the use of the scanning transmission electron microscopy (STEM). Tomography analyses using STEM of thick sections from yeast and mammalian cells are of higher quality than are bright-field (BF) images. In this study, we compared regular BF tomograms and STEM tomograms from 500-nm thick sections from hypertrophied Golgi stacks of alfalfa root cap cells. Due to their thickness and intense heavy metal staining, BF tomograms of the thick sections suffer from poor contrast and high noise levels. We were able to mitigate these drawbacks by using STEM tomography. When we performed STEM tomography of densely stained chloroplasts of Arabidopsis cotyledon, we observed similar improvements relative to BF tomograms. A longer time is required to collect a STEM tilt series than similar BF TEM images, and dynamic autofocusing required for STEM imaging often fails at high tilt angles. Despite these limitations, STEM tomography is a powerful method for analyzing structures of large or dense organelles of plant cells.

Quantifying morphological features of actin cytoskeletal filaments in plant cells based on mathematical morphology

By quantifying the morphological properties of biological structures, we can better evaluate complex shapes and detect subtle morphological changes in organisms. In this paper, we propose a shape analysis method based on morphological image processing, and apply it to image analysis of actin cytoskeletal filaments in root hair cells of Arabidopsis thaliana. In plant cells, the actin cytoskeletal filaments have critical roles in various cellular processes such as vesicle trafficking and organelle motility. The dynamics of vesicles and organelles in plant cells depend on actin cytoskeletal filaments, regulating cell division and cell enlargement. To better understand the actin-dependent organelle motility, we attempted to quantify the organization of actin filaments in the root hair cells of the root hair defective 3 (rhd3) mutant. RHD3 is involved in actin organization, and its defect has been reported to affect the dynamics of various vesicles and organelles. We measured three shape features of the actin filaments in wild-type and mutant plants. One feature (thickness) was depicted on a grayscale; the others (describing the complexity of the filament network patterns in two-dimensional space) were depicted as binary features. The morphological phenotypes of the cytoskeletal filaments clearly differed between wild-type and mutant. Subtle variations of filament morphology among the mutants were detected and statistically quantified.

New Trends on Optical Fiber Tweezers

In the last few decades, optical trapping has played an unique role concerning contactless trapping and manipulation of biological specimens. More recently, optical fiber tweezers (OFTs) are emerging as a desirable alternative to bulk optical systems. In this paper, an overview of the state of the art of OFTs is presented, focusing on the main fabrication methods, their features and main achievements. In addition, new OFTs fabricated by guided wave photo polymerization are reported. Their theoretical and experimental characterization is given and results demonstrating its application in the manipulation of yeast cells and the organelles of plant cells are presented.

A Bioimaging Pipeline to Show Membrane Trafficking Regulators Localized to the Golgi Apparatus and Other Organelles in Plant Cells

A Bioimaging Pipeline to Show Membrane Trafficking Regulators Localized to the Golgi Apparatus and Other Organelles in Plant Cells

Intracellular localization, accumulation and distribution of heavy metals in plants

Heavy metals uptake, distribution and accumulation processes in plants are very important for their impact on physiological and biochemical processes and, consequently, on plant growth and development. The distribution of heavy metals in plant parts and cell organelles are discussed. There are detoxification mechanisms in cell compartments, like binding with cell wall, with organic acids in vacuoles, complexes with phytochelatins and etc. What proportion of given metal ions would be the in free form, and what – bound with organic molecules; it depends from pH of the environment and chemical properties of element. The stability of metals complexes decreases in the case of deviation pH of environment from neutral: at low pH there is a competition of protons with metal ions for binding sites in molecules, at high pH – by the reason of the competition of hydroxyl groups with ligand.

Variation and stability in chloroplast behavior of green-gram genotypes through streptomycin-induced bleaching

Chloroplasts are the important plant cell organelles. To study the variation in chloroplast behavior through streptomycin induced bleaching, nine green-gram genotypes were treated with 500, 1000 and 2000 ppm of aqueous streptomycin (SM) solution. Variation in chloroplast behavior was measured in terms of bleaching index (BI). Green-gram genotypes differed in BI values indicating variation in their chloroplast behavior. BI value showed positive correlation (0.708) with yielding ability. The genotypes formed two clusters following the numerical taxonomic approach. The consistency in chloroplast behavior that indirectly indicates adaptation pattern of the genotypes was predicted using relative bleaching response index (RBRI).

Polyamines are common players in different facets of plant programmed cell death.

Programmed cell death (PCD) is a process that occurs throughout the life span of every plant life, from initial germination of the seed to the senescence of the plant. It is a normal physiological milestone during the plant's developmental process, but it can also be induced by external factors, including a variety of environmental stresses and as a response to pathogen infections. Changes in the morphology of the nucleus is one of the most noticeable during PCD but all the components of the plant cell (cytoplasm, cytoskeleton and organelles) are involved in this fascinating process. To date, relatively little is known about PCD in plants, but several factors, among which polyamines (PAs) and plant growth regulators, have been shown to play an important role in the initiation and regulation of the process. The role of PAs in plant PCD appears to be multifaceted acting in some instances as pro-survival molecules, whereas in others seem to be implicated in accelerating PCD. The molecular mechanism is still under study. Here we present some PCD plant models, focusing on the role of the enzyme responsible for PA conjugation to proteins: transglutaminase (TGase), an enzyme linked with the process of PCD also in some animal models. The role of PAs and plant TGase in the senescence and PCD in flowers, leaf and the self-incompatibility of pollen will be discussed and examined in depth.

Vacuolar Sorting Receptor-Mediated Trafficking of Soluble Vacuolar Proteins in Plant Cells.

Vacuoles are one of the most prominent organelles in plant cells, and they play various important roles, such as degradation of waste materials, storage of ions and metabolites, and maintaining turgor. During the past two decades, numerous advances have been made in understanding how proteins are specifically delivered to the vacuole. One of the most crucial steps in this process is specific sorting of soluble vacuolar proteins. Vacuolar sorting receptors (VSRs), which are type I membrane proteins, are involved in the sorting and packaging of soluble vacuolar proteins into transport vesicles with the help of various accessory proteins. To date, large amounts of data have led to the development of two different models describing VSR-mediated vacuolar trafficking that are radically different in multiple ways, particularly regarding the location of cargo binding to, and release from, the VSR and the types of carriers utilized. In this review, we summarize current literature aimed at elucidating VSR-mediated vacuolar trafficking and compare the two models with respect to the sorting signals of vacuolar proteins, as well as the molecular machinery involved in VSR-mediated vacuolar trafficking and its action mechanisms.

Plant Vacuolar Trafficking Occurs through Distinctly Regulated Pathways

The multifunctional vacuole is the largest organelle in plant cells, and many proteins are transported to and stored in this organelle; thus, the vacuole has great physiological and agronomical importance. However, the molecular mechanism and regulation of plant vacuolar traffic remain largely unknown. In this study, we demonstrate that multiple vacuolar trafficking pathways operate in plants. RAB5 and RAB7 are evolutionarily conserved subfamilies of Rab GTPase, whose animal and yeast counterparts regulate vacuolar/endosomal trafficking in a sequential manner. Functional analyses of a putative activating complex for RAB7 indicated that this complex is responsible for maturation from RAB5- to RAB7-positive endosomes in plant cells. Moreover, these machinery components are recruited to a more complex trafficking network. Mutations in RAB5 and RAB7 conferred counteracting effects on the vti11 mutant. Furthermore, impairment of RAB5- and RAB7-dependent pathways differentially affected the transport of distinctive cargos. These results indicate that plants have developed a complex vacuolar transport system distinct from that of nonplant systems by assigning evolutionarily conserved machinery to unique trafficking pathways. These pathways provide a fundamental basis for plant development at the cellular and higher-ordered levels.

Exploring ligand recognition, selectivity and dynamics of TPR domains of chloroplast Toc64 and mitochondria Om64 fromArabidopsis thaliana

The study aims to gain insight into the mode of ligand recognition by tetratricopeptide repeat (TPR) domains of chloroplast translocon at the outer envelope of chloroplast (Toc64) and mitochondrial Om64, two paralogous proteins that mediate import of proteins into chloroplast and mitochondria, respectively. Chaperone proteins associate with precursor proteins in the cytosol to maintain them in a translocation competent conformation and are recognized by Toc64 and Om64 that are located on the outer membrane of the target organelle. Heat shock proteins (Hsp70) and Hsp90 are two chaperones, which are known to play import roles in protein import. The C-termini of these chaperones are known to interact with the TPR domain of chloroplast Toc64 and mitochondrial Om64 in Arabidopsis thaliana (At). Using a molecular dynamics approach and binding energy calculations, we identify important residues involved in the interactions. Our findings suggest that the TPR domain from AtToc64 has higher affinity towards C-terminal residues of Hsp70. The interaction occurs as the terminal helices move towards each other enclosing the cradle on interaction of AtHsp70 with the TPR domain. In contrast, the TPR domain from AtOm64 does not discriminate between the C-termini of Hsp70 and Hsp90. These binding affinities are discussed with respect to our knowledge of protein targeting and specificity of protein import into endosymbiotic organelles in plant cells.

Evolutionary Development of Redox Regulation in Chloroplasts

The post-translational modification of thiol groups stands out as a key strategy that cells employ for metabolic regulation and adaptation to changing environmental conditions. Nowhere is this more evident than in chloroplasts-the O2-evolving photosynthetic organelles of plant cells that are fitted with multiple redox systems, including the thioredoxin (Trx) family of oxidoreductases functional in the reversible modification of regulatory thiols of proteins in all types of cells. The best understood member of this family in chloroplasts is the ferredoxin-linked thioredoxin system (FTS) by which proteins are modified via light-dependent disulfide/dithiol (S-S/2SH) transitions. Discovered in the reductive activation of enzymes of the Calvin-Benson cycle in illuminated chloroplast preparations, recent studies have extended the role of the FTS far beyond its original boundaries to include a spectrum of cellular processes. Together with the NADP-linked thioredoxin reductase C-type (NTRC) and glutathione/glutaredoxin systems, the FTS also plays a central role in the response of chloroplasts to different types of stress. The comparisons of redox regulatory networks functional in chloroplasts of land plants with those of cyanobacteria-prokaryotes considered to be the ancestors of chloroplasts-and different types of algae summarized in this review have provided new insight into the evolutionary development of redox regulation, starting with the simplest O2-evolving organisms. The evolutionary appearance, mode of action, and specificity of the redox regulatory systems functional in chloroplasts, as well as the types of redox modification operating under diverse environmental conditions stand out as areas for future study.

Organelle-localized potassium transport systems in plants

Some intracellular organelles found in eukaryotes such as plants have arisen through the endocytotic engulfment of prokaryotic cells. This accounts for the presence of plant membrane intrinsic proteins that have homologs in prokaryotic cells. Other organelles, such as those of the endomembrane system, are thought to have evolved through infolding of the plasma membrane. Acquisition of intracellular components (organelles) in the cells supplied additional functions for survival in various natural environments. The organelles are surrounded by biological membranes, which contain membrane-embedded K+ transport systems allowing K+ to move across the membrane. K+ transport systems in plant organelles act coordinately with the plasma membrane intrinsic K+ transport systems to maintain cytosolic K+ concentrations. Since it is sometimes difficult to perform direct studies of organellar membrane proteins in plant cells, heterologous expression in yeast and Escherichia coli has been used to elucidate the function of plant vacuole K+ channels and other membrane transporters. The vacuole is the largest organelle in plant cells; it has an important task in the K+ homeostasis of the cytoplasm. The initial electrophysiological measurements of K+ transport have categorized three classes of plant vacuolar cation channels, and since then molecular cloning approaches have led to the isolation of genes for a number of K+ transport systems. Plants contain chloroplasts, derived from photoautotrophic cyanobacteria. A novel K+ transport system has been isolated from cyanobacteria, which may add to our understanding of K+ flux across the thylakoid membrane and the inner membrane of the chloroplast. This chapter will provide an overview of recent findings regarding plant organellar K+ transport proteins.

Live imaging and image analysis of organelles in plant cells(<Feature Articles>Live-cell imaging)

Live imaging and image analysis of organelles in plant cells(<Feature Articles>Live-cell imaging)