A new tool for plant cell biology: in vivo antibody uptake in plant protoplasts

We report on the in vivo uptake of antibodies into plant protoplasts. When protoplasts of sunflower, Arabidopsis or tobacco were incubated in vivo with an antibody, this antibody was detected by immunofluorescence in the cytoplasm and/or the nucleus, depending on the location of the target protein. Furthermore, when protoplasts were cultured in the presence of antibodies, specific effects were observed. Incubation with antibodies raised against p34^cdc2 led to a strong inhibition of the division rate, and a decrease in the average DNA content of protoplasts. With antibodies against HaWLIM1, a LIM domain protein of the CRP type, a negative effect on actin organisation was observed. We conclude that antibodies can penetrate plant protoplasts in vivo, and thus may be used as powerful tools for the study of protein function.     

Modification-specific proteomics in plant biology

Post-translational modifications (PTMs) are involved in the regulation of a wide range of biological processes, and affect e.g. protein structure, activity and stability. Several hundred PTMs have been described in the literature, but relatively few have been studied using mass spectrometry and proteomics. In general, methods for PTM characterization are developed to study yeast and mammalian biology and later adopted to investigate plants. Our point of view is that it is advantageous to enrich for PTMs on the peptide level as part of a quantitative proteomics strategy to not only identify the PTM, but also to determine the functional relevance in the context of regulation, response to abiotic stress etc. Protein phosphorylation is the only PTM that has been studied extensively at the proteome wide level in plants using mass spectrometry based methods.     

Circular RNA in cardiovascular disease: Expression,mechanisms and clinical prospects

Circular RNAs (circRNAs) are a group of covalently closed, endogenous, non-coding RNAs, which exist widely in human tissues including the heart. Increasing evidence has shown that cardiac circRNAs play crucial regulatory roles in cardiovascular diseases (CVDs). In this review, we aimed to provide a systemic understanding of circRNAs with a special emphasis on the cardiovascular system. We have summarized the current research on the classification, biogenesis and properties of circRNAs as well as their participation in the pathogenesis of CVDs. CircRNAs are conserved, stable and have specific spatiotemporal expression; thus, they have been accepted as a potential diagnostic marker or an incremental prognostic biomarker for CVDs.     

Intertidal rocky shore biotopes of the Azores: a quantitative approach

Rocky littoral communities of the Azores are dominated by macroalgae where turf formations represent the main life form as on many subtropical and tropical shores. The present study aimed at describing and classifying benthic intertidal biotopes of the Azores using clear quantification and data analysis methodologies to be used in similar studies for spatial and/or temporal comparison. This numerical approach intends to have management and conservation applicability. Thirty study locations along the coast of São Miguel island were characterised by substratum type (cobbles, boulders or bedrock – different stability levels) and exposure to wave action (high, medium or low exposure), from June to August 2002. Algae, sessile and sedentary invertebrates were recorded along transects, and the more conspicuous taxa quantified. Community structure is mainly shaped by substratum stability – unstable cobble communities are separate from those of more stable boulders and bedrock. Boulders present an intermediate community composition between cobbles and bedrock. Exposure to wave action induces smaller variation in community composition. Sixteen biotopes have been recognised. Ultimately the present paper provides an objective set of biotope definition tools for a broad public whose interest focuses on spatial and temporal comparison of coastal communities and associated habitats.     

Regulation of plant alternative oxidase activity: A tale of two cysteines

Two Cys residues, Cys_I and Cys_II, are present in most plant alternative oxidases (AOXs). Cys_I inactivates AOX by forming a disulfide bond with the corresponding Cys_I residue on the adjacent subunit of the AOX homodimer. When reduced, Cys_I associates with α-keto acids, such as pyruvate, to activate AOX, an effect mimicked by charged amino acid substitutions at the Cys_I site. Cys_II may also be a site of AOX activity regulation, through interaction with the small α-keto acid, glyoxylate. Comparison of Arabidopsis AOX1a (AtAOX1a) mutants with single or double substitutions at Cys_I and Cys_II confirmed that glyoxylate interacted with either Cys, while the effect of pyruvate (or succinate for AtAOX1a substituted with Ala at Cys_I) was limited to Cys_I. A variety of Cys_II substitutions constitutively activated AtAOX1a, indicating that neither the catalytic site nor, unlike at Cys_I, charge repulsion is involved. Independent effects at each Cys were suggested by lack of Cys_II substitution interference with pyruvate stimulation at Cys_I, and close to additive activation at the two sites. However, results obtained using diamide treatment to covalently link the AtAOX1a subunits by the disulfide bond indicated that Cys_I must be in the reduced state for activation at Cys_II to occur.     

Proteome of plant peroxisomes: new perspectives on the role of these organelles in cell biology

Peroxisomes are cell organelles bounded by a single membrane with a basically oxidative metabolism. Peroxisomes house catalase and H₂O₂‐producing flavin‐oxidases as the main protein constituents. However, since their discovery in early fifties, a number of new enzymes and metabolic pathways have been reported to be also confined to these organelles. Thus, the presence of exo‐ and endo‐peptidases, superoxide dismutases, the enzymes of the plant ascorbate‐glutathione cycle plus ascorbate and glutathione, several NADP‐dehydrogenases, and also L‐arginine‐dependent nitric oxide synthase activity has evidenced the relevant role of these organelles in cell physiology. In recent years, the study of new functions of peroxisomes has become a field of intensive research in cell biology, and these organelles have been proposed to be a source of important signal molecules for different transduction pathways. In plants, peroxisomes participate in seed germination, leaf senescence, fruit maturation, response to abiotic and biotic stress, photomorphogenesis, biosynthesis of the plant hormones jasmonic acid and auxin, and in cell signaling by reactive oxygen and nitrogen species (ROS and RNS, respectively). In order to decipher the nature and specific role of the peroxisomal proteins in these processes, several approaches including in vivo and in vitro import assays and generation of mutants have been used. In the last decade, the development of genomics and the report of the first plant genomes provided plant biologists a powerful tool to assign to peroxisomes those proteins which harbored any of the two peroxisomal targeting signals (PTS, either PTS1 or PTS2) described so far. Unfortunately, those molecular approaches could not give any response to those proteins previously localized in plant peroxisomes by classical biochemical and cell biology methods that did not contain any PTS. However, more recently, proteomic studies of highly purified organelles have provided evidence of the presence in peroxisomes of new proteins not previously reported. Thus, the contribution of proteomic approaches to the biology of peroxisomes is essential, not only for elucidation of the mechanisms involved in the import of the PTS1‐ and PTS2‐independent proteins, but also to the understanding of the role of these organelles in the cell physiology of plant growth and development.     

Kinetic and regulatory aspects of the function of the alternative oxidase in plant respiration

The kinetic modelling of the respiratory network in plant mitochondria is discussed, with emphasis on the importance of the choice of boundary conditions, and of modelling of both quinol-oxidising and quinone-reducing pathways. This allows quantitative understanding of the interplay between the different pathways, and of the functioning of the plant respiratory network in terms of the kinetic properties of its component parts. The effects of activation of especially succinate dehydrogenase and the cyanide-insensitive alternative oxidase are discussed. Phenomena, such as respiratory control ratios depending on the substrate, shortcomings of the Bahr and Bonner model for electron distribution between the oxidases and reversed respiratory control, are explained. The relation to metabolic control analysis of the respiratory network is discussed in terms of top-down analysis.     

A comparison of the LDL-cholesterol lowering efficacy of plant stanols and plant sterols over a continuous dose range: results of a meta-analysis of randomized, placebo-controlled trials

Purpose

To determine if plant stanols and plant sterols differ with respect to their low-density lipoprotein cholesterol (LDL-CH) lowering efficacies across a continuous dose range.

Methods

Dose-response relationships were evaluated separately for plant stanols and plant sterols and reductions in LDL-CH, using a first-order elimination function.

Results

Altogether, 113 publications and 1 unpublished study report (representing 182 strata) complied with the pre-defined inclusion and exclusion criteria and were included in the assessment. The maximal LDL-CH reductions for plant stanols (16.4%) and plant stanol ester (17.1%) were significantly greater than the maximal LDL-CH reductions for plant sterols (8.3%) and plant sterol ester (8.4%). These findings persisted in several additional analyses.

Discussion and conclusions

Intakes of plant stanols in excess of the recommended 2 g/day dose are associated with additional and dose-dependent reductions in LDL-CH, possibly resulting in further reductions in the risk of coronary heart disease (CHD).     

Reconstructing ancient environments: A role for fossils in biology courses

A role is suggested for geological sites in ecology courses for school and college students. From observations on the nature of the rock and, particularly, its fossils it is possible to deduce the nature of the environment at the time the rock was deposited. An example of a checklist for use in the field shows the kind of observations and deductions that can be made on rock structures, textures, fossil morphology, taxonomy, and mode of preservation. Notes are provided on means by which fossils may be extracted and cleaned for examination and identification. The final section deals with the problem of finding suitable sites and the appropriate literature for identifying and interpreting the material found.     

Betalain production in plant in vitro systems

Betalains have been widely used as natural colorants for many centuries, but their attractiveness for use as colorants of foods (or drugs and cosmetics) has increased recently due to their reportedly high anti-oxidative, free radical scavenging activities and concerns about the use of various synthetic alternatives. The main commercial sources of betalains are powders and concentrates of red beet (Beta vulgaris) or cactus pear (Opuntia ficus-indica) extracts. However, in recent years the technical and commercial feasibility of various in vitro systems to produce them biotechnologically has been explored. These research activities have included assessments of novel approaches for cultivating plant cell or tissue cultures, and diverse bioreactor systems for increasing production levels of secondary metabolites. This paper reviews recent progress in plant in vitro systems for producing betalain pigments. In addition, the factors that could be manipulated, the bioreactor systems that could be used, and the strategies that could be applied to improve betalain production are discussed.     

Modification of plant hormone levels and signaling as a tool in plant biotechnology

Plant hormones are signal molecules, present in trace quantities, that act as major regulators of plant growth and development. They are involved in a wide range of processes such as elongation, flowering, root formation and vascular differentiation. For many years, agriculturists have applied hormones to their crops to either increase the yield, or improve the quality of the commercial product. Nowadays, the knowledge of hormone biosynthesis, degradation and signaling pathways has allowed the utilization of biotechnological tools to further improve the main agricultural crops. Natural or artificial mutants, with impaired functioning of the corresponding genes, have been adopted because of their superior phenotype in specific agricultural traits. In addition, transgenic plants have been generated to regulate internal hormone levels, or their signaling pathways, resulting in some crops that have revolutionized agriculture.     

Identification of intra- and intermolecular disulphide bonding in the plant mitochondrial proteome by diagonal gel electrophoresis

Redox active proteins in plant mitochondria were examined using 2-D oxidant/reductant diagonal-SDS-PAGE to separate and identify proteins with intermolecular or intramolecular disulphide bonds using diamide in the first dimension and DTT in the second dimension. Eighteen proteins spots were resolved either above or below the diagonal and these were in-gel digested and identified by MS/MS. This analysis revealed intermolecular disulphide bonds in alternative oxidase, O-acetylserine (thiol) lyase, citrate synthase and between subunits of the ATP synthase. Intramolecular disulphide bonds were observed in a range of mitochondrial dehydrogenases, elongation factor Tu, adenylate kinase and the phosphate translocator. Many of the soluble proteins found were known glutaredoxin/thioredoxin targets in other plants, but the membrane proteins were not found by these methods nor were the nature of the disulphides able to be investigated. The accessibility of thiols involved in disulphide bonds to modification by a lipid derived aldehyde gave an insight into the potential impact of Cys modification on redox-functions in mitochondria during lipid peroxidation. Comparison of the protein sequences of the identified proteins with homologs from other species has identified specific Cys residues that may be responsible for plant-specific redox modulations of mitochondrial proteins.