Development of a K(+)-channel probe and its use for identification of an intracellular plant membrane K+ channel.

Polyclonal antibodies were generated against a 9-amino acid, synthetic peptide corresponding to the selectivity filter in the pore region of K(+)-channel proteins. The sequence of amino acids in the ion-conducting pore region of K+ channels is the only highly conserved region of members of this protein family. The objectives of the present work were (i) to determine whether the anti-channel pore peptide antibody was immunoreactive with known K(+)-channel proteins and (ii) to demonstrate the usefulness of the antibody by employing it to identify a newly discovered K(+)-channel protein. Anti-channel pore peptide was immunoreactive with various K(+)-channel subtypes native to a number of different species. Immunoblot analysis demonstrated affinity of the antibody for the drk1, maxi-K, and KAT1 K(+)-channel proteins. Studies also suggested that the anti-channel pore peptide antibody did not immunoreact with membrane proteins other than K+ channels. The anti-channel pore peptide antibody was used to establish the identity of a 62-kDa chloroplast inner envelope polypeptide as a putative component of a K(+)-channel protein. It was concluded that an antibody generated against the conserved pore region/selectivity filter of K+ channels has broad but selective affinity for this class of proteins. This K(+)-channel probe may be a useful tool for identification of K(+)-channel proteins in native membranes.

Componentes genéticos que afetam a via de direcionamento de proteínas organelares em Arabidopsis thaliana

Nos eucariotos, a evolução dos sistemas de transporte molecular foi essencial pois seu alto grau de compartimentalização requer mecanismos com maior especificidade para a localização de proteínas. Com o estabelecimento das mitocôndrias e plastídeos como organelas da célula eucariota, grande parte dos genes específicos para sua atividade e manutenção foram transferidos ao núcleo. Após a transferência gênica, a maioria das proteínas passaram a ser codificadas pelo núcleo, sintetizadas no citosol e direcionadas às organelas por uma maquinaria complexa que envolve receptores nas membranas das organelas, sequências de direcionamento nas proteínas e proteínas citossólicas que auxiliam o transporte. A importação depende em grande parte de uma sequência na região N-terminal das proteínas que contém sinais reconhecidos pelas membranas organelares. No entanto, muito ainda não é compreendido sobre o transporte de proteínas organelares e fatores ainda desconhecidos podem influenciar o direcionamento sub-celular. O objetivo deste trabalho foi a caracterização da General Regulatory Factor 9 (GRF9), uma proteína da família 14-3-3 de Arabidopsis thaliana potencialmente envolvida no direcionamento de proteínas organelares, e a geração de um genótipo para ser utilizado na obtenção de uma população mutante para genes que afetam o direcionamento da proteína Tiamina Monofosfato Sintetase (TH-1). Após experimentos in vivo e in planta, foi observado que GRF9 interage com as proteínas duplo-direcionadas Mercaptopyruvate Sulfurtransferase1 (MST1) e a Thiazole Biosynthetic Enzyme (THI1), e com a proteína direcionada aos cloroplastos TH-1. Experimentos de deleção e interação in vivo mostraram que a região Box1 de GRF9 é essencial para a interação com THI1 e MST1. Com a finalidade de dar continuidade a caracterização da GRF9 e para realização de testes com relação a sua função no direcionamento de proteínas organelares foi gerada uma linhagem homozigota que superexpressa GRF9. Plantas expressando o transgene TH-1 fusionado a Green Fluorescent Protein (GFP) em genótipo deficiente na TH-1 (CS3469/TH-1-GFP) foram obtidas para a geração de população mutante que possibilitará a descoberta de componentes genéticos ainda desconhecidos e responsáveis pelo direcionamento de proteínas aos cloroplastos.

Biosystematics of the Genus Andryala L. (Asteraceae)

Andryala (Asteraceae: Cichorieae) is a little-known Mediterranean-Macaronesian genus whose taxonomy is much in need of revision. The aim of the present biosystematic study was to elucidate species relationships within this genus based on morphological and molecular data. In this study several taxa are recognised: 17 species, 14 subspecies, and 3 hybrids. Among these, 5 species are Macaronesian endemics (A. glandulosa, A. sparsiflora, A. crithmifolia Aiton, A. pinnatifida, and A. perezii), 4 species are Northwest African endemics (A. mogadorensis, A. maroccana, A. chevallieri, and A. nigricans) and one species is endemic to Romania (A. laevitomentosa). Historical background regarding taxonomic delimitation in the genus is addressed from Linnaean to present day concepts, as well as the origin of the name Andryala. The origin of Asteraceae and the systematic position of Andryala is shortly summarised. The morphological study was based on a bibliographic review and the revision of 1066 specimens of 13 herbaria as well as additional material collected during fieldwork. The variability of the morphological characters of the genus, including both vegetative taxonomic characters (root, stem, leaf and indumentum characters) and reproductive ones (inflorescence, floret, fruit and pappus characters), is assessed. Numerical analysis of the morphological data was performed using different similarity or dissimilarity measures and coefficients, as well as ordination and clustering methods. Results support the segregation of the recognised taxa and the congruence of the several analyses in the separation of the recognised taxa (using quantitative, binary or multi-state characters). The proposed taxonomy for Andryala includes a new infra-generic classification, new taxa and new combinations and ranks, typifications and diagnostic keys (one for the species and several for subspecies). For each taxon a list of synonyms, typification comments and a detailed description are provided, just as comments on taxonomy and nomenclature, and a brief discussion on karyology. Additionally, information on ecology and conservation status as well as on distribution and a list of studied material are also presented. Phylogenetic analyses based on different nuclear and chloroplast DNA markers, using Bayesian and maximum parsimony methods of inference, were performed. Results support three main lineages: separate ones for the relict species A. agardhii and A. laevitomentosa and a third including the majority of the Andryala species that underwent a relatively rapid and recent speciation. They also suggest a single colonization event of Madeira and the Canary Islands from the Mediterranean region, followed by insular speciation. Biogeography and speciation within the genus are briefly discussed, including a proposal for the centre of origin of the genus and possible dispersal routes.

Influence of nitrogen deficiency on senescence and the amounts of RNA and proteins in wheat leaves

Senescence-associated coordination in amounts of enzymes localized in different cellular compartments were determined in attached leaves of young wheat (Triticum aestivum L. cv. Arina) plants. Senescence was initiated at the time of full leaf elongation based on declines in total RNA and soluble protein. Removal of N from the growth medium just at the time of full leaf elongation enhanced the rate of senescence. Sustained declines in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), and a marked decrease in the rbcS transcripts, just after full leaf elongation indicated that Rubisco synthesis/degradation was very sensitive to the onset of senescence. Rubisco activase amount also declined during senescence but the proportion of rca transcript relative to the total poly A RNA pool increased 3-fold during senescence. Thus, continued synthesis of activase may be required to maintain functional Rubisco throughout senescence. N stress led to declines in the amount of proteins located in the chloroplast, the peroxisome and the cytosol. Transcripts of the Clp protease subunits also declined in response to N stress, indicating that Clp is not a senescence-specific protease. In contrast to the other proteins, mitochondrial NADH-glutamate dehydrogenase (EC 1.4.1.2) was relatively stable during senescence and was not affected by N stress. During natural senescence with adequate plant nitrate supply the amount of nitrite reductase (EC 1.7.7.1) increased, and those of glutamine synthetase (EC 1.4.7.1) and glutamate synthase (EC 6.3.1.2) were stable. These results indicated that N assimilatory capacity can continue or even increase during senescence if the substrate supply is maintained. Differential stabilities of proteins, even within the same cellular compartment, indicate that proteolytic activity during senescence must be highly regulated.

Knock-out of Arabidopsis metal transporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects

IRT1 and IRT2 are members of the Arabidopsis ZIP metal transporter family that are specifically induced by iron deprivation in roots and act as heterologous suppressors of yeast mutations inhibiting iron and zinc uptake. Although IRT1 and IRT2 are thought to perform redundant functions as root-specific metal transporters, insertional inactivation of the IRT1 gene alone results in typical symptoms of iron deficiency causing severe leaf chlorosis and lethality in soil. The irt1 mutation is characterized by specific developmental defects, including a drastic reduction of chloroplast thylakoid stacking into grana and lack of palisade parenchyma differentiation in leaves, reduced number of vascular bundles in stems, and irregular patterns of enlarged endodermal and cortex cells in roots. Pulse labeling with 59Fe through the root system shows that the irt1 mutation reduces iron accumulation in the shoots. Short-term labeling with 65Zn reveals no alteration in spatial distribution of zinc, but indicates a lower level of zinc accumulation. In comparison to wild-type, the irt1 mutant responds to iron and zinc deprivation by altered expression of certain zinc and iron transporter genes, which results in the activation of ZIP1 in shoots, reduction of ZIP2 transcript levels in roots, and enhanced expression of IRT2 in roots. These data support the conclusion that IRT1 is an essential metal transporter required for proper development and regulation of iron and zinc homeostasis in Arabidopsis.

Total V9 rDNA information organized at the metabarcode level (Database W4)

The present data set provides a tab separated text file compressed in a zip archive. The file includes metadata for each TaraOceans V9 rDNA metabarcode including the following fields: md5sum = unique identifier; lineage = taxonomic path associated to the metabarcode; pid = % identity to the closest reference barcode from V9_PR2; sequence = nucleotide sequence of the metabarcode; refs = identity of the best hit reference sequence(s); TARA_xxx = number of occurrences of this barcode in each of the 334 samples; totab = total abundance of the barcode ; cid = identifier of the OTU to which the barcode belongs; and taxogroup = high-taxonomic level assignation of this barcode. The file also includes three categories of functional annotations: (1) Chloroplast: yes, presence of permanent chloroplast; no, absence of permanent chloroplast ; NA, undetermined. (2) Symbiont (small partner): parasite, the species is a parasite; commensal, the species is a commensal; mutualist, the species is a mutualist symbiont, most often a microalgal taxon involved in photosymbiosis; no the species is not involved in a symbiosis as small partner; NA, undetermined. (3) Symbiont (host): photo, the host species relies on a mutualistic microalgal photosymbiont to survive (obligatory photosymbiosis); photo_falc, same as photo, but facultative relationship; photo_klep, the host species maintains chloroplasts from microalgal prey(s) to survive; photo_klep_falc, same as photo_klep, but facultative; Nfix, the host species must interact with a mutualistic symbiont providing N2 fixation to survive; Nfix_falc, same as Nfix, but facultative; no, the species is not involved in any mutualistic symbioses; NA, undetermined. For example, the collodarian/Brandtodinium symbiosis is annotated: Chloroplast, "no"; Symbiont (small), "no"; Symbiont (host), "photo", for the collodarian host; and: Chloroplast, "yes"; Symbiont (small), "mutualist"; Symbiont (host), "no", for the dinoflagellate microalgal endosymbiont.chloroplast = "yes", "no" or "NA"; symbiont.small = "parasite", "commensal", "mutualist", "no" or "NA"; symbiont.host = "photo", "photo_falc", "photo_klep", "Nfix", no or NA; benef = "Nfix", "no" or "NA"; trophism = Metazoa , heterotroph , NA , photosymbiosis , phototroph according to the previous fields.

Highly organized structure in the non-coding region of the psbA minicircle from clade C Symbiodinium

The chloroplast genes of dinoflagellates are distributed among small, circular dsDNA molecules termed minicircles. In this paper, we describe the structure of the non-coding region of the psbA minicircle from Symbiodinium. DNA sequence was obtained from five Symbiodinium strains obtained from four different coral host species (Goniopora tenuidens, Heliofungia actiniformis, Leptastrea purpurea and Pocillopora damicornis), which had previously been determined to be closely related using LSU rDNA region D1/D2 sequence analysis. Eight distinct sequence blocks, consisting of four conserved cores interspersed with two metastable regions and flanked by two variable regions, occurred at similar positions in all strains. Inverted repeats (IRs) occurred in tandem or 'twin' formation within two of the four cores. The metastable regions also consisted of twin IRs and had modular behaviour, being either fully present or completely absent in the different strains. These twin IRs are similar in sequence to double-hairpin elements (DHEs) found in the mitochondrial genomes of some fungi, and may be mobile elements or may serve a functional role in recombination or replication. Within the central unit (consisting of the cores plus the metastable regions), all IRs contained perfect sequence inverses, implying they are highly evolved. IRs were also present outside the central unit but these were imperfect and possessed by individual strains only. A central adenine-rich sequence most closely resembled one in the centre of the non-coding part of Amphidinium operculatum minicircles, and is a potential origin of replication. Sequence polymorphism was extremely high in the variable regions, suggesting that these regions may be useful for distinguishing strains that cannot be differentiated using molecular markers currently available for Symbiodinium.

Targeting genetic resource conservation in widespread species: a case study of Cedrela odorata L.

Conservation of genetic resources is a recognised necessity for the long term maintenance of evolutionary potential. Effective assessment and implementation Strategies are required to permit rapid evaluation and protection of resources. Here we use information from the chloroplast, total genome and quantitative characters assayed across wide-ranging populations to assess genetic resources in a Neotropical tree, Cedrela odorata. A major differentiation identified for organelle, total genomic and quantitative variation was found to coincide with an environmental gradient across Costa Rica. However, a major evolutionary divergence between the Yucatan region and Honduras/Nicaragua identified within the chloroplast genome was not differentiated using quantitative characters. Based on these and other results, a three-tiered conservation genetic prioritisation process is recommended. In order of importance, and where information is available, conservation units should be defined using quantitative (expressed genes), nuclear (genetic connectivity) and organellar (evolutionary) measures. Where possible, information from range wide and local scale studies should be combined and emphasis should be placed on coincidental disjunctions for two or more measures. However, if only rapid assessments of diversity are possible, then assessment of organelle variation provides the most cautious assessment of genetic resources, at least for C. odorata, and can be used to propose initial conservation units. When considering effective implementation of genetic resource management strategies a final tier should be considered, that of landuse/geopolitical divisions. (C) 2004 Elsevier B.V. All rights reserved.

Genetic effects of chronic habitat fragmentation on tree species: the case of Sorbus aucuparia in a deforested Scottish landscape

Sustainable forest restoration and management practices require a thorough understanding of the influence that habitat fragmentation has on the processes shaping genetic variation and its distribution in tree populations. We quantified genetic variation at isozyme markers and chloroplast DNA (cpDNA), analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in severely fragmented populations of Sorbus aucuparia (Rosaceae) in a single catchment (Moffat) in southern Scotland. Remnants maintain surprisingly high levels of gene diversity (H-E) for isozymes (H-E = 0.195) and cpDNA markers (H-E = 0.490). Estimates are very similar to those from non-fragmented populations in continental Europe, even though the latter were sampled over a much larger spatial scale. Overall, no genetic bottleneck or departures from random mating were detected in the Moffat fragments. However, genetic differentiation among remnants was detected for both types of marker (isozymes Theta(n) = 0.043, cpDNA Theta(c) = 0.131; G-test, P-value < 0.001). In this self-incompatible, insect-pollinated, bird-dispersed tree species, the estimated ratio of pollen flow to seed flow between fragments is close to 1 (r = 1.36). Reduced pollen-mediated gene flow is a likely consequence of habitat fragmentation, but effective seed dispersal by birds is probably helping to maintain high levels of genetic diversity within remnants and reduce genetic differentiation between them.

High nuclear genetic diversity, high levels of outcrossing and low differentiation among remnant populations of Quercus petraea at the margin of its range in Ireland

Background and Aims Quercus petraea colonized Ireland after the last glaciation from refugia on mainland Europe. Deforestation. however. beginning in Neolithic times, has resulted in small, scattered forest fragments, now covering less than 12 000 ha. Methods Plastid (three fragments) and microsatellite variation (13 loci) were characterized in seven Irish populations sampled along a north-south gradient. Using Bayesian approaches and Wright's F-statistics, the effects of colonization and fragmentation on the genetic structure and mating patterns of extant oak populations were investigated. Key-Results All Populations possessed cytotypes common to the Iberian Peninsula. Despite the distance from the refugial core and the extensive deforestation in Ireland, nuclear genetic variation was high and comparable to mainland Europe. Low population differentiation was observed within Ireland and populations showed no evidence for isolation by distance. As expected of a marker with an effective Population size of one-quarter relative to the nuclear genome, plastid variation indicated higher differentiation. Individual inbreeding coefficients indicated high levels of outcrossing. Conclusions Consistent with a large effective Population size in the historical migrant gene pool and/or with high gene flow among populations, high within-population diversity and low population differentiation was observred within Ireland. It is proposed that native Q. petraea populations in Ireland share a common phylogeographic history and that the present genetic structure does not reflect founder effects. (C) 2004 Annals of Botany Company.

An investigation into effects of long-distance seed dispersal on organelle population genetic structure and colonization rate: a model analysis

A simulation-based modelling approach is used to examine the effects of stratified seed dispersal (representing the distribution of the majority of dispersal around the maternal parent and also rare long-distance dispersal) on the genetic structure of maternally inherited genomes and the colonization rate of expanding plant populations. The model is parameterized to approximate postglacial oak colonization in the UK, but is relevant to plant populations that exhibit stratified seed dispersal. The modelling approach considers the colonization of individual plants over a large area (three 500 km x 10 km rolled transects are used to approximate a 500 km x 300 km area). Our approach shows how the interaction of plant population dynamics with stratified dispersal can result in a spatially patchy haplotype structure. We show that while both colonization speeds and the resulting genetic structure are influenced by the characteristics of the dispersal kernel, they are robust to changes in the periodicity of long-distance events, provided the average number of long-distance dispersal events remains constant. We also consider the effects of additional physical and environmental mechanisms on plant colonization. Results show significant changes in genetic structure when the initial colonization of different haplotypes is staggered over time and when a barrier to colonization is introduced. Environmental influences on survivorship and fecundity affect both the genetic structure and the speed of colonization. The importance of these mechanisms in relation to the postglacial spread and genetic structure of oak in the UK is discussed.

The decomposition of starch grains in soils: implications for archaeological residue analyses

Recent research involving starch grains recovered from archaeological contexts has highlighted the need for a review of the mechanisms and consequences of starch degradation specifically relevant to archaeology. This paper presents a review of the plant physiological and soil biochemical literature pertinent to the archaeological investigation of starch grains found as residues on artefacts and in archaeological sediments. Preservative and destructive factors affecting starch survival, including enzymes, clays, metals and soil properties, as well as differential degradation of starches of varying sizes and amylose content, were considered. The synthesis and character of chloroplast-formed 'transitory' starch grains, and the differentiation of these from 'storage' starches formed in tubers and seeds were also addressed. Findings of the review include the higher susceptibility of small starch grains to biotic degradation, and that protective mechanisms are provided to starch by both soil aggregates and artefact surfaces. These findings suggest that current reasoning which equates higher numbers of starch grains on an artefact than in associated sediments with the use of the artefact for processing starchy plants needs to be reconsidered. It is argued that an increased understanding of starch decomposition processes is necessary to accurately reconstruct both archaeological activities involving starchy plants and environmental change investigated through starch analysis. (C) 2004 Elsevier Ltd. All rights reserved.

Monitoring genetic diversity in tropical trees with multilocus dominant markers

Since no universal codominant markers are currently available, dominant genetic markers, such as amplified fragment length polymorphism (AFLP), are valuable tools for assessing genetic diversity in tropical trees. However, the measurement of genetic diversity (H) with dominant markers depends on the frequency of null homozygotes (Q) and the fixation index (F) of populations. While Q can be estimated for AFLP loci, F is less accessible. Through a modelling approach, we show that the monolocus estimation of genetic diversity is strongly dependent on the value of F, but that the multilocus diversity estimate is surprisingly robust to variations in F. The robustness of the estimate is due to a mechanistic effect of compensation between negative and positive biases of H by different AFLP loci exhibiting contrasting frequency profiles of Q. The robustness was tested across contrasting theoretical frequency profiles of Q and verified for 10 neotropical species. Practical recommendations for the implementation of this analytical method are given for genetic surveys in tropical trees, where such markers are widely applied.

Improved photobiological H-2 production in engineered green algal cells

Oxygenic photosynthetic organisms use solar energy to split water (H2O) into protons (H+), electrons (e(-)), and oxygen. A select group of photosynthetic microorganisms, including the green alga Chlamydomonas reinhardtii, has evolved the additional ability to redirect the derived H+ and e(-) to drive hydrogen (H-2) production via the chloroplast hydrogenases HydA1 and A2 (H(2)ase). This process occurs under anaerobic conditions and provides a biological basis for solar-driven H-2 production. However, its relatively poor yield is a major limitation for the economic viability of this process. To improve H-2 production in Chlamydomonas, we have developed a new approach to increase H+ and e(-) supply to the hydrogenases. In a first step, mutants blocked in the state 1 transition were selected. These mutants are inhibited in cyclic e(-) transfer around photosystem I, eliminating possible competition for e(-) with H(2)ase. Selected strains were further screened for increased H-2 production rates, leading to the isolation of Stm6. This strain has a modified respiratory metabolism, providing it with two additional important properties as follows: large starch reserves ( i.e. enhanced substrate availability), and a low dissolved O-2 concentration (40% of the wild type (WT)), resulting in reduced inhibition of H2ase activation. The H-2 production rates of Stm6 were 5 - 13 times that of the control WT strain over a range of conditions ( light intensity, culture time, +/- uncoupler). Typically, similar to 540 ml of H-2 liter(-1) culture ( up to 98% pure) were produced over a 10-14-day period at a maximal rate of 4 ml h(-1) ( efficiency = similar to 5 times the WT). Stm6 therefore represents an important step toward the development of future solar-powered H-2 production systems.