Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif

Several macrocyclic peptides (similar to 30 amino acids), with diverse biological activities, have been isolated from the Rubiaceae and Violaceae plant families over recent years. We have significantly expanded the range of known macrocyclic peptides with the discovery of 16 novel peptides from extracts of Viola hederaceae, Viola odorata and Oldenlandia affinis. The Viola plants had not previously been examined for these peptides and thus represent novel species in which these unusual macrocyclic peptides are produced. Further, we have determined the three-dimensional struc ture of one of these novel peptides, cycloviolacin O1, using H-1 NMR spectroscopy. The structure consists of a distorted triple-stranded beta-sheet and a cystine-knot arrangement of the disulfide bonds. This structure is similar to kalata B1 and circulin A, the only two macrocyclic peptides for which a structure was available, suggesting that despite the sequence variation throughout the peptides they form a family in which the overall fold is conserved. We refer to these peptides as the cyclotide family and their embedded topology as the cyclic cystine knot (CCK) motif. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals they can be separated into two subfamilies, one of which tends to contain a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-Pro peptide bond and may conceptually be regarded as a molecular Moebius strip. Here we define the structural features of the two apparent subfamilies of the CCK peptides which may be significant for the likely defense related role of these peptides within plants. (C) 1999 Academic Press.

Implications of specimen preparation and of surface contamination for the measurement of the grain boundary carbon concentration of steels using x-ray microanalysis in an UHVFESTEM

The purpose of the present investigation was to gain an understanding of the nature of the carbon contamination on the surface of standard steel transmission electron spectroscopy (TEM) specimens, the effect of exposure of a clean specimen to normal laboratory air, and the efficacy of plasma-cleaning treatments. This knowledge is a necessary prerequisite to the development of appropriate specimen preparation and/or specimen cleaning methods. X-ray photoelectron spectroscopy in combination with argon ion beam profiling was used to characterize the specimen surfaces of X65 steel and 316 stainless steel. The only clean carbon-free surface obtained was that during argon etching of the sample in the surface analysis chamber. Any exposure of a previously cleaned sample to laboratory air resulted in a rapid carbon (hydrocarbon) contamination of the sample surface and the development of surface oxidation, Plasma cleaning with subsequent exposure of the specimen to the laboratory air also resulted in a carbon-contaminated surface. This suggests that procedures of preparation of TEM specimens of steels outside an ultrahigh vacuum chamber are unlikely to result in the lowering of contamination rates on specimens to levels where measurements for carbon in the grain boundaries are possible. What is needed is a cleaning system as an integral part of the specimen insertion system into the field-emission scanning transmission electron microscope. This cleaning could be carried out by argon ion etching. Copyright (C) 2000 John Wiley & Sons, Ltd.

X-ray absorption and phase contrast imaging to study the interplay between plant roots and soil structure

Plant performance is, at least partly, linked to the location of roots with respect to soil structure features and the micro-environment surrounding roots. Measurements of root distributions from intact samples, using optical microscopy and field tracings have been partially successful but are imprecise and labour-intensive. Theoretically, X-ray computed micro-tomography represents an ideal solution for non-invasive imaging of plant roots and soil structure. However, before it becomes fast enough and affordable or easily accessible, there is still a need for a diagnostic tool to investigate root/soil interplay. Here, a method for detection of undisturbed plant roots and their immediate physical environment is presented. X-ray absorption and phase contrast imaging are combined to produce projection images of soil sections from which root distributions and soil structure can be analyzed. The clarity of roots on the X-ray film is sufficient to allow manual tracing on an acetate sheet fixed over the film. In its current version, the method suffers limitations mainly related to (i) the degree of subjectivity associated with manual tracing and (ii) the difficulty of separating live and dead roots. The method represents a simple and relatively inexpensive way to detect and quantify roots from intact samples and has scope for further improvements. In this paper, the main steps of the method, sampling, image acquisition and image processing are documented. The potential use of the method in an agronomic perspective is illustrated using surface and sub-surface soil samples from a controlled wheat trial. Quantitative characterization of root attributes, e.g. radius, length density, branching intensity and the complex interplay between roots and soil structure, is presented and discussed.

Coordinated plant defense responses in Arabidopsis revealed by microarray analysis

Disease resistance is associated with a plant defense response that involves an integrated set of signal transduction pathways. Changes in the expression patterns of 2.375 selected genes were examined simultaneously by cDNA microarray analysis in Arabidopsis thaliana after inoculation with an incompatible fungal pathogen Alternaria brassicicola or treatment with the defense-related signaling molecules salicylic acid (SA), methyl jasmonate (MJ), or ethylene, Substantial changes (up- and down-regulation) in the steady-state abundance of 705 mRNAs were observed in response to one or more of the treatments, including known and putative defense-related genes and 106 genes with no previously described function or homology, In leaf tissue inoculated with A. brassicicola, the abundance of 168 mRNAs was increased more than 2.5-fold, whereas that of 39 mRNAs was reduced. Similarly, the abundance of 192, 221, and 55 mRNAs was highly (>2.5-fold) increased after treatment with SA, MJ, and ethylene, respectively. Data analysis revealed a surprising level of coordinated defense responses, including 169 mRNAs regulated by multiple treatments/defense pathways. The largest number of genes coinduced (one of four induced genes) and corepressed was found after treatments with SA and MJ. In addition, 50% of the genes induced by ethylene treatment were also induced by MJ treatment. These results indicated the existence of a substantial network of regulatory interactions and coordination occurring during plant defense among the different defense signaling pathways, notably between the salicylate and jasmonate pathways that were previously thought to act in an antagonistic fashion.

A method for setting the size of plant conservation target areas

Realistic time frames in which management decisions are made often preclude the completion of the detailed analyses necessary for conservation planning. Under these circumstances, efficient alternatives may assist in approximating the results of more thorough studies that require extensive resources and time. We outline a set of concepts and formulas that may be used in lieu of detailed population viability analyses and habitat modeling exercises to estimate the protected areas required to provide desirable conservation outcomes for a suite of threatened plant species. We used expert judgment of parameters and assessment of a population size that results in a specified quasiextinction risk based on simple dynamic models The area required to support a population of this size is adjusted to take into account deterministic and stochastic human influences, including small-scale disturbance deterministic trends such as habitat loss, and changes in population density through processes such as predation and competition. We set targets for different disturbance regimes and geographic regions. We applied our methods to Banksia cuneata, Boronia keysii, and Parsonsia dorrigoensis, resulting in target areas for conservation of 1102, 733, and 1084 ha, respectively. These results provide guidance on target areas and priorities for conservation strategies.

Movement-related potentials in Huntington's disease: movement preparation and execution

Movement-related potentials (MRPs) reflect increasing cortical activity related to the preparation and execution of voluntary movement. Execution and preparatory components may be separated by comparing MRPs recorded from actual and imagined movement. Imagined movement initiates preparatory processes, but not motor execution activity. MRPs are maximal over the supplementary motor area (SMA), an area of the cortex involved in the planning and preparation of movement. The SMA receives input from the basal ganglia, which are affected in Huntington's disease (HD), a hyperkinetic movement disorder. In order to further elucidate the effects of the disorder upon the cortical activity relating to movement, MRPs were recorded from ten HD patients, and ten age-matched controls, whilst they performed and imagined performing a sequential button-pressing task. HD patients produced MRPs of significantly reduced size both for performed and imagined movement. The component relating to movement execution was obtained by subtracting the MRP for imagined movement from the MRP for performed movement, and was found to be normal in HD. The movement preparation component was found by subtracting the MRP found for a control condition of watching the visual cues from the MRP for imagined movement. This preparation component in HD was reduced in early slope, peak amplitude, and post-peak slope. This study therefore reported abnormal MRPs in HD. particularly in terms of the components relating to movement preparation, and this finding may further explain the movement deficits reported in the disease.

An L-system-based plant modeling language

Cpfg is a program for simulating and visualizing plant development, based on the theory of L-systems. A special-purpose programming language, used to specify plant models, is an essential feature of cpfg. We review postulates of L-system theory that have influenced the design of this language. We then present the main constructs of this language, and evaluate it from a user's perspective.

The preparation and execution of self-initiated and externally-triggered movement: A study of event-related fMRI

Studies of functional brain imaging in humans and single cell recordings in monkeys have generally shown preferential involvement of the medially located supplementary motor area (SMA) in self-initiated movement and the lateral premotor cortex in externally cued movement. Studies of event-related cortical potentials recorded during movement preparation, however, generally show increased cortical activity prior to self-initiated movements but little activity at early stages prior to movements that are externally cued at unpredictable times. In this study, the spatial location and relative timing of activation for self-initiated and externally triggered movements were examined using rapid event-related functional MRI. Twelve healthy right-handed subjects were imaged while performing a brief finger sequence movement (three rapid alternating button presses: index-middle-index finger) made either in response to an unpredictably timed auditory cue (between 8 to 24 s after the previous movement) or at self-paced irregular intervals. Both movement conditions involved similar strong activation of medial motor areas including the pre-SMA, SMA proper, and rostral cingulate cortex, as well as activation within contralateral primary motor, superior parietal, and insula cortex. Activation within the basal ganglia was found for self-initiated movements only, while externally triggered movements involved additional bilateral activation of primary auditory cortex. Although the level of SMA and cingulate cortex activation did not differ significantly between movement conditions, the timing of the hemodynamic response within the pre-SMA was significantly earlier for self-initiated compared with externally triggered movements. This clearly reflects involvement of the pre-SMA in early processes associated with the preparation for voluntary movement. (C) 2002 Elsevier Science.

Macro-botanical evidence for plant use at Neolithic Catalhoyuk, south-central Anatolia, Turkey

Analysis of charred plant macro-remains, including wood charcoals, cereals, seeds, tubers and fruits from the Neolithic site of Catalhoyuk has indicated complex patterns of plant resource use and exploitation in the Konya plain during the early Holocene. Evidence presented in this paper shows that settlement location was not dictated by proximity to high quality arable land and direct access to arboreal resources (firewood, timber, fruit producing species). A summary of the patterns observed in sample composition and species representation is outlined here together with preliminary interpretations of these results within their broader regional context.

Surface reflectance properties of Antarctic moss and their relationship to plant species, pigment composition and photosynthetic function

In this study the variations in surface reflectance properties and pigment concentrations of Antarctic moss over species, sites, microtopography and with water content were investigated. It was found that species had significantly different surface reflectance properties, particularly in the region of the red edge (approximately 700 nm), but this did not correlate strongly with pigment concentrations. Surface reflectance of moss also varied in the visible region and in the characteristics of the red edge over different sites. Reflectance parameters, such as the photochemical reflectance index (PRI) and cold hard band were useful discriminators of site, microtopographic position and water content. The PRI was correlated both with the concentrations of active xanthophyll-cycle pigments and the photosynthetic light use efficiency, F-v/F-m, measured using chlorophyll fluorescence. Water content of moss strongly influenced the amplitude and position of the red-edge as well as the PRI, and may be responsible for observed differences in reflectance properties for different species and sites. All moss showed sustained high levels of photoprotective xanthophyll pigments, especially at exposed sites, indicating moss is experiencing continual high levels of photochemical stress.

Plant growth limitation and nutrient loss following piled burning in slash and burn agriculture

In the forest zone of Cameroon, small-scale family farmers practicing traditional slash and burn practices achieve a clear field by piled burning of the branches and trunks of cleared vegetation. Plant growth inhibition on ash patches, and the risk of nutrient loss from these areas, was evaluated on field plots on which 0.5 t m(-2) or 1.0 t m(-2) of wood was piled and burnt, and in laboratory studies. The ash produced by burning was strongly alkaline, and laboratory bio-assessment studies showed that the saline, high pH conditions produced in ash patches prevented germination and plant growth for up to two wet seasons, as is observed in the field. Field and laboratory studies demonstrated rapid release (1 wet season) of K and S from the ash and the loss of a substantial portion of these nutrients from the soil profile by leaching. In contrast, leaching carries Mg from the ash gradually (3 to 4 wet seasons), while Ca, Cu, Zn and P are leached slowly. The nutrients contained in ash patches are considered at risk of loss both through leaching (K and S) and by erosion of ash (Ca, Mg, Cu, Zn and P). Farmers should be encouraged to spread ash patches prior to cultivation in order to exploit the nutrient content of ash and to lessen the risk of nutrient loss.

The preparation and readiness for voluntary movement: a high-field event-related fMRI study of the Bereitschafts-BOLD response

Activity within motor areas of the cortex begins to increase 1 to 2 s prior to voluntary self-initiated movement (termed the Bereitschaftspotential or readiness potential). There has been much speculation and debate over the precise source of this early premovement activity as it is important for understanding the roles of higher order motor areas in the preparation and readiness for voluntary movement. In this study, we use high-field (3-T) event-related fMRI with high temporal sampling (partial brain volumes every 250 ms) to specifically examine hemodynamic response time courses during the preparation, readiness, and execution of purely self-initiated voluntary movement. Five right-handed healthy volunteers performed a rapid sequential finger-to-thumb movement performed at self-determined times (12-15 trials). Functional images for each trial were temporally aligned and the averaged time series for each subject was iteratively correlated with a canonical hemodynamic response function progressively shifted in time. This analysis method identified areas of activation without constraining hemodynamic response timing. All subjects showed activation within frontal mesial areas, including supplementary motor area (SMA) and cingulate motor areas, as well as activation in left primary sensorimotor areas. The time courses of hemodynamic responses showed a great deal of variability in shape and timing between subjects; however, four subjects clearly showed earlier relative hemodynamic responses within SMA/cingulate motor areas compared with left primary motor areas. These results provide further evidence that the SMA and cingulate motor areas are major contributors to early stage premovement activity and play an important role in the preparation and readiness for voluntary movement. (C) 2003 Elsevier Inc. All rights reserved.

Simultaneous X-ray imaging of plant root growth and water uptake in thin-slab systems

Direct and simultaneous observation of root growth and plant water uptake is difficult because soils are opaque. X-ray imaging techniques such as projection radiography or Computer Tomography (CT) offer a partial alternative to such limitations. Nevertheless, there is a trade-off between resolution, large field-of-view and 3-dimensionality: With the current state of the technology, it is possible to have any two. In this study, we used X-ray transmission through thin-slab systems to monitor transient saturation fields that develop around roots as plants grow. Although restricted to 2-dimensions, this approach offers a large field-of-view together with high spatial and dynamic resolutions. To illustrate the potential of this technology, we grew peas in 1 cm thick containers filled with soil and imaged them at regular intervals. The dynamics of both the root growth and the water content field that developed around the roots could be conveniently monitored. Compared to other techniques such as X-ray CT, our system is relatively inexpensive and easy to implement. It can potentially be applied to study many agronomic problems, such as issues related to the impact of soil constraints (physical, chemical or biological) on root development.

A comparison of the self-association behavior of the plant cyclotides kalata B1 and kalata B2 via analytical ultracentrifugation

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted.We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mM, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe(6), and the second involving a charge-charge interaction with the Asp(25) residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules.