In planta analysis of protein–protein interactions related to light signaling by bimolecular fluorescence complementation

The determination of protein-protein interactions is becoming more and more important in the molecular analysis of signal transduction chains. To this purpose the application of a manageable and simple assay in an appropriate biological system is of major concern. Bimolecular fluorescence complementation (BiFC) is a novel method to analyze protein-protein interactions in vivo. The assay is based on the observation that N- and C-terminal subfragments of the yellow-fluorescent protein (YFP) can only reconstitute a functional fluorophore when they are brought into tight contact. Thus, proteins can be fused to the YFP subfragments and the interaction of the fusion proteins can be monitored by epifluorescence microscopy. Pairs of interacting proteins were tested after transient cotransfection in etiolated mustard seedlings, which is a well characterized plant model system for light signal transduction. BiFC could be demonstrated with the F-box protein EID1 (empfindlicher im dunkelroten Licht 1) and the Arabidopsis S-phase kinase-related protein 1 (ASK1). The interaction of both proteins was specific and strictly dependent on the presence of an intact F-box domain. Our studies also demonstrate that etiolated mustard seedlings provide a versatile transient assay system to study light-induced subcellular localization events.     

Induced resistance in the indeterminate growth of aspen (<Emphasis Type="Italic">Populus tremuloides</Emphasis>)

Studies of induction in trees have examined rapid induced resistance (RIR) or delayed induced resistance (DIR), but have not examined induction that occurs in leaves produced by indeterminately growing trees subsequent to, but in the same season as, damage. We refer to induction that occurs during this time period as intermediate-delayed induced resistance (IDIR). We assessed the influences of genetic and environmental factors, and their interactions, on temporal and spatial variation in induction and on tradeoffs between induced and constitutive levels of resistance in indeterminately growing saplings of aspen (Populus tremuloides). We utilized a common garden of 12 aspen genotypes experiencing two levels of defoliation and two levels of soil nutrients. We assessed concentrations of phenolic glycosides and condensed tannins in damaged leaf remnants collected 1 week after defoliation to examine rapid and local induction, and in undamaged leaves produced 8 weeks after defoliation to assess intermediate-delayed and systemic induction. In general, tannins showed RIR, while phenolic glycosides expressed IDIR. For both classes of allelochemicals, we found high estimates of broad-sense heritability and genetic variation in both induced and constitutive levels. Genetic variation may be maintained by both direct costs of allelochemicals and by costs of inducibility (phenotypic plasticity). Such costs may drive the tradeoff exhibited between induced and constitutive levels of phenolic glycosides. IDIR may be important in reducing total-season tissue loss by providing augmented resistance against late summer herbivores in trees that have experienced damage earlier in the season. Herbivore-resistant compensatory growth is especially beneficial to young trees growing in competitive environments.     

Binding and transfer of an oligodeoxynucleotide containing the translation initiation site of the <Emphasis Type="Italic">BCL2</Emphasis> mRNA into K562 cells

A study was conducted of the interaction of an octadecameric oligodeoxynucleotide (dON) containing the BCL2 mRNA translation start with K562 cells. Both in solution and upon lipofection, the binding of dON used at a low concentration (30 nM) at 37°C involved two steps: saturating surface binding with the cell membrane and internalization. Three phases were revealed in the dynamics of internalization: the extent and rate of internalization increased during the first hour of incubation; decreased during the second hour; and then increased again, which was assumed to reflect the priming of new dON-binding sites. The binding constant and the number of binding sites were estimated at 10°C (the conditions preventing internalization) by consecutive dissociation of dON-cell complexes formed in 1 h at 37°C. Incubation of dON with cells led to the priming of new high-affinity binding sites and an increase of the binding constant to a level characteristic of high-affinity ligand-receptor interactions (10⁹ M^–1). High-affinity receptor-mediated binding preceded internalization of dON. Lipofection increased the binding constant and the number of binding sites severalfold but had virtually no effect on the temporal pattern and the extent of dON internalization.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 235–244.Original Russian Text Copyright © 2005 by Timofeev, Borovkova, Nydenova, Akhlynina, Shmarov, Grineva.     

Statistical convenience vs biological insight: consequences of data transformation for the analysis of fitness variation in heterogeneous environments

In plants, more favourable environmental conditions can lead to dramatic increases in both mean fitness and variance in fitness. This results in data that violate the equality-of-variance assumption of anova, a problem that most empiricists would address by log-transforming fitness values. Using heuristic data sets and simple simulations, we show that anova on log-transformed fitness consistently fails to match the outcome of selection in a heterogeneous environment or its sensitivity to environmental frequency. Only anova based on relative fitness within environments accurately predicts the sensitivity of genotype selection to the frequency of alternative environments. Parallel analyses of variance based on absolute fitness and relative fitness can bracket the expected success of alternative genotypes under hard and soft selection, respectively. For example, for Sinapis arvensis growing in full sun and partial shade treatments, families achieving high fitness in the best environment are favoured under hard selection, whereas soft selection favours different families that achieve consistently good performance across environments. Based on these findings, we recommend that log-transformation of fitness should no longer be standard practice in ecological genetics studies. Weighted anova is a preferable method for dealing with unequal variances, and investigators should also make greater use of techniques such as quantile regression or resampling to describe and evaluate fitness variation across heterogeneous environments.     

The Vh8 locus of a new gene-for-gene interaction between Venturia inaequalis and the wild apple Malus sieversii is closely linked to the Vh2 locus in Malus pumila R12740-7A

The wild apple (Malus sieversii) is a large-fruited species from Central Asia, which is used as a source of scab resistance in cultivar breeding. Phytopathological tests with races of Venturia inaequalis were performed to differentiate scab-resistance genes in Malus as well as an avirulence gene in the pathogen. A novel gene-for-gene interaction between V. inaequalis and Malus was identified. The locus of the scab-resistance gene Vh8 is linked with, or possibly allelic to, that of the Vh2 gene in Malus pumila Russian apple R12740-7A, at the lower end of linkage group 2 of Malus. Race 8 isolate NZ188B.2 is compatible with Vh8, suggesting the loss or modification of the complementary AvrVh8 gene, while isolate 1639 overcomes both Vh2 and Vh8, but is incompatible with at least one other gene not detected by any of the other race isolates tested. Our research is the first to differentiate scab-resistance genes in a putative gene cluster in apple with the aid of races of V. inaequalis.     

Infection of Chinese cabbage by Plasmodiophora brassicae leads to a stimulation of plant growth: impacts on cell wall metabolism and hormone balance

The importance of plant hormones in clubroot infection has long been recognized. The morphological changes, such as cell division and cell elongation leading to gall formation are triggered in the early stages of infection. We analysed cell expansion by localizing Xyloglucan endoTransglucosylase/Hydrolase (XTH)-action and screened the endogenous concentrations of several classes of phytohormones by mass spectrometry in the early stages of Plasmodiophora brassicae infection in Chinese cabbage (Brassica rapa spp. pekinensis). Infected plants showed a general transient growth promotion early in infection. Furthermore a clear XTH action was visible in the epidermal layer of infected roots. Complex changes in the endogenous phytohormone profile were observed. Initially infection resulted in an increased total auxin pool. The auxin increase, together with an increased XTH action, results in wall loosening and consequently cell expansion. When the first secondary plasmodia are formed, thirteen days after infection (DAI), can be considered a switch point in phytohormone metabolism. Twenty-one DAI the plasmodia might act as a plant hormone sink resulting in a reduction in the active cytokinin pool and a lower indole-3-acetic acid content in the infected plants.     

Systemic response to aphid infestation by <Emphasis Type="Italic">Myzus persicae</Emphasis> in the phloem of <Emphasis Type="Italic">Apium graveolens</Emphasis>

Little is known about the molecular processes involved in the phloem response to aphid feeding. We investigated molecular responses to aphid feeding on celery (Apium graveolenscv. Dulce) plants infested with the aphid Myzus persicae, as a means of identifying changes in phloem function. We used celery as our model species as it is easy to separate the phloem from the surrounding tissues in the petioles of mature leaves of this species. We generated a total of 1187 expressed sequence tags (ESTs), corresponding to 891 non-redundant genes. We analysed these ESTs in silico after cDNA macroarray hybridisation. Aphid feeding led to significant increase in RNA accumulation for 126 different genes. Different patterns of deregulation were observed, including transitory or stable induction 3 or 7days after infestation. The genes affected belonged to various functional categories and were induced systemically in the phloem after infestation. In particular, genes involved in cell wall modification, water transport, vitamin biosynthesis, photosynthesis, carbon assimilation and nitrogen and carbon mobilisation were up-regulated in the phloem. Further analysis of the response in the phloem or xylem suggested that a component of the response was developed more specifically in the phloem. However, this component was different from the stress responses in the phloem driven by pathogen infection. Our results indicate that the phloem is actively involved in multiple adjustments, recruiting metabolic pathways and in structural changes far from aphid feeding sites. However, they also suggest that the phloem displays specific mechanisms that may not be induced in other tissues.EST, macroarray and clustering data are available from our website [http://www-biocel.versailles.inra.fr/phloem]. Data deposition: The sequences reported in this paper have been deposited in the Genbank database (Accession nos.: AY607692-AY607700, AY611007, CN253939-CN255151, CV512445-CV512447 and CV651120-CV651121).     

恢复及演替过程中的土壤生态学考虑

 人类社会的日益扩张,导致人类加速占据地球表面景观,并胁迫地球上生态系统提供不断增长的资源需求和废物吸收能力。所以保护尚未“开放”的自然生态系统及恢复退化的生态系统成为人类长期生存的重要保证。该文着重讨论了恢复过程中的土壤生态学问题。土壤是所有陆地生态系统的结构与功能基础。土壤微生物与动物的种群变化,土壤有机质的积累,及主要元素地球化学循环的改变是恢复生态的重要环节。生态恢复与演替有许多共性,所以演替理论对于认识生态系统恢复中的结构与功能变化有着很大帮助。与自然演替不同的是,人的积极参与在生态恢复中占有中心位置。从最初样地的确立与物种的选择,到后续的灌溉与施肥管理,人的选择影响着土壤的演化,生态系统的发展方向,和最终恢复生态的结果。为保障恢复生态系统的可持续性,短期的工作目标,如提供养分促进植物生长,务必与长期的工作目标,如土壤的恢复相结合。植物与土壤的相互反馈是生态恢复成功的重要标志。成功的生态恢复不仅是对现有生态学理论的“试金检验”,也是推动生态学学科发展的重要原动力。