Evaluation of stem injection for managing giant reed (Arundo donax).

Giant reed is an emergent aquatic plant that may be weedy in riparian habitats. Two herbicides approved for controlling giant reed in the US are glyphosate (N-(phosphonomethyl) glycine) and imazapyr (2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridinecarboxylic acid). Foliar applications of these herbicides may be restricted in some areas, such as those, which are within the range of threatened or endangered species. We conducted two field experiments at sites in northern and central California. The first experiment evaluated the effects of three aquatic herbicides (glyphosate, imazapyr, and triclopyr [(3,5,6-trichloro-2-pyridinyl)oxy]acetic acid) injected into all of the stems within a giant reed (5 mL stem(-1)). In this experiment, leaf chlorophyll content, the proportion of living stems, and the number of new stems produced during the year after treatment declined (>80%) following injection of either full strength glyphosate or imazapyr. The effects of injecting full strength triclopyr were considerably less. In a second experiment, different proportions (0, 10%, 25%, or 100%) of the stems within a plant were injected with full strength glyphosate. Results indicated that it was necessary to inject all of the stems within a clump to achieve the greatest reduction in the plant growth characteristics measured. These results imply that giant reed may be successfully controlled by injecting full strength glyphosate (5 mL stem(-1)) into all of the stems within a clump. While labor intensive and thus potentially more costly this method, offers a new method for managing giant reed in sensitive sites where foliar spray applications may be restricted.

Proteomics reveals plastid- and periplastid-targeted proteins in the chlorarachniophyte alga Bigelowiella natans.

Chlorarachniophytes are unicellular marine algae with plastids (chloroplasts) of secondary endosymbiotic origin. Chlorarachniophyte cells retain the remnant nucleus (nucleomorph) and cytoplasm (periplastidial compartment, PPC) of the green algal endosymbiont from which their plastid was derived. To characterize the diversity of nucleus-encoded proteins targeted to the chlorarachniophyte plastid, nucleomorph, and PPC, we isolated plastid-nucleomorph complexes from the model chlorarachniophyte Bigelowiella natans and subjected them to high-pressure liquid chromatography-tandem mass spectrometry. Our proteomic analysis, the first of its kind for a nucleomorph-bearing alga, resulted in the identification of 324 proteins with 95% confidence. Approximately 50% of these proteins have predicted bipartite leader sequences at their amino termini. Nucleus-encoded proteins make up >90% of the proteins identified. With respect to biological function, plastid-localized light-harvesting proteins were well represented, as were proteins involved in chlorophyll biosynthesis. Phylogenetic analyses revealed that many, but by no means all, of the proteins identified in our proteomic screen are of apparent green algal ancestry, consistent with the inferred evolutionary origin of the plastid and nucleomorph in chlorarachniophytes.

Ribosomal RNA genes in Euglena gracilis mitochondrial DNA: fragmented genes in a seemingly fragmented genome.

Because relatively little information is available about mtDNA in the euglenid protozoa, distant relatives of the kinetoplastid protozoa, we investigated mitochondrial genome structure and expression in Euglena gracilis. We found that isolated E. gracilis mtDNA comprises a heterodisperse collection of short molecules (modal size approximately 4 kbp) and that the mitochondrial large subunit (LSU) and small subunit (SSU) rRNAs are each split into two pieces. For the two halves of the SSU rRNA, we identified separate, non-contiguous coding modules that are flanked by a complex array of (primarily direct) A + T-rich repeats. The potential secondary structure of the bipartite SSU rRNA displays the expected conserved elements implicated in ribosome function. Label from [α-(32)P]GTP was incorporated in the presence of guanylyltransferase into each of the separate SSU and LSU rRNA fragments, confirming that these RNAs are primary transcripts, separately expressed from non-contiguous rRNA modules. In addition to authentic genes for SSU rRNA, we discovered numerous short fragments of protein-coding and rRNA genes dispersed throughout the E. gracilis mitochondrial genome. We propose that antisense transcripts of gene fragments of this type could have been the evolutionary precursors of the guide RNAs that mediate U insertion/deletion editing in the kinetoplastid relatives of the euglenids. To the extent that E. gracilis mtDNA is a representative euglenid mitochondrial genome, it differs radically in structure and organization from that of its kinetoplastid relatives, instead more closely resembling the mitochondrial genome of dinoflagellates in many of its features, an apparent evolutionary convergence.

Generation and analysis of expressed sequence tags from the medicinal plant Salvia miltiorrhiza.

Salvia miltiorrhiza Bge. is a well-known traditional Chinese herb. Its roots have been formulated and used clinically for the treatment of various diseases. However, little genetic information has so far been available and this fact has become a major obstacle for molecular studies. To address this lack of genetic information, an Expressed Sequence Tag (EST) library from whole plantlets of S. miltiorrhiza was generated. From the 12959 cDNA clones that were randomly selected and subjected to single-pass sequencing from their 5' ends, 10288 ESTs (with sizes > or = 100 bp) were selected and assembled into 1288 contigs, leaving 2937 singletons, for a total of 4225 unigenes. These were analyzed using BLASTX (against protein databases), RPS-BLAST (against a conserved domain database) as well as the web-based KEGG Automatic Annotation Server for metabolic enzyme assignment. Based on the metabolic enzyme assignment, expression patterns of 14 secondary metabolic enzyme genes in different organs and under different treatments were verified using real-time PCR analysis. Additionally, a total of 122 microsatellites were identified from the ESTs, with 89 having sufficient flanking sequences for primer design. This set of ESTs represents a significant proportion of the S. miltiorrhiza transcriptome, and gives preliminary insights into the gene complement of S. miltiorrhiza. They will prove useful for uncovering secondary metabolic pathways, analyzing cDNA-array based gene expression, genetic manipulation to improve yield of desirable secondary products, and molecular marker identification.

Canopy assessment of biochemical features by ground-based hyperspectral data for an invasive species, giant reed (Arundo donax).

This study explored the potential use of hyperspectral data in the non-destructive assessment of chlorophyll, carbon, and nitrogen content of giant reed at the canopy level. We found that pseudoabsorption and derivatives of original hyperspectral data were able to describe the relationship between spectral data and measured biochemical characteristics. Based on correlogram analyses of ground-based hyperspectral data, we found that derivatives of pseudoabsorption were the best predictors of chlorophyll, carbon, and nitrogen content of giant reed canopies. Within the visible region, spectral data significantly correlated with chlorophyll content at both 461 nm and 693 nm wavelengths. Within the near-infrared region, carbon levels correlated with hyperspectral data at five causal wavelengths: 1038 nm, 1945 nm, 1132 nm, 1525 nm, and 1704 nm. The best spectral wavelength for estimating nitrogen content was 1542 nm. Such relationships between nutrient content and spectral data were best represented by exponential functions in most situations.

Exploring the mitochondrial proteome of the ciliate protozoon Tetrahymena thermophila: direct analysis by tandem mass spectrometry.

To date, direct analysis of mitochondrial proteomes has largely been limited to animals, fungi and plants. To broaden our knowledge of mitochondrial structure and function, and to provide additional insight into the evolution of this key eukaryotic organelle, we have undertaken the first comprehensive analysis of the mitochondrial proteome of a protist. Highly purified mitochondria from Tetrahymena thermophila, a ciliated protozoon, were digested exhaustively with trypsin and the resulting peptides subjected to tandem liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS). In this way, we directly identified a total of 573 mitochondrial proteins, 545 of which are encoded by the nuclear genome and 28 by the mitochondrial genome. The latter number includes a novel, 44 residue protein (which we designate Ymf78) that had not been recognized during annotation of the T. thermophila mtDNA sequence. The corresponding gene, ymf78, is highly conserved in genomic position, size and sequence within the genus Tetrahymena. Our analysis has provided broad coverage of both membrane-bound and soluble proteins from the various submitochondrial compartments, with prominent representatives including components of the tricarboxylic acid cycle, Complexes I-IV of the electron transport chain and Complex V (ATP synthase), the mitochondrial transcription and translation machinery, the TOM and TIM protein translocases, various mitochondrial transporters, chaperonins (Cpn60, Hsp70, Hsp90), at least four FtsH family ATP-dependent metalloproteases implicated in m-AAA and i-AAA protease function, and enzymes involved in lipid, amino acid and coenzyme metabolism, as well as iron-sulfur cluster formation. Unexpectedly, six of the ten enzymes of glycolysis were found by MS analysis of purified T. thermophila mitochondria, whereas no hits were seen to any cytosolic ribosomal proteins. At least one of the glycolytic proteins, enolase, has an evident N-terminal extension that exhibits characteristics of a typical mitochondrial targeting peptide. As in other organisms, phylogenetic analysis of functionally annotated mitochondrial proteins demonstrates that <20% can be traced confidently to the alpha-proteobacterial lineage of Bacteria, emphasizing the chimeric evolutionary nature of the mitochondrial proteome. Notably, about 45% of the proteins identified in our analysis have no known function, and most of these do not have obvious homologs outside of the ciliate lineage. About two-thirds of these ORFan proteins have putative homologs in another ciliate, Paramecium tetraurelia, whereas the remainder appear to be Tetrahymena-specific. These results emphasize the power and importance of direct MS-based analysis of mitochondria in revealing novel mitochondrial proteins in different eukaryotic lineages. Our observations reinforce an emerging view of the mitochondrion as an evolutionarily flexible organelle, with novel proteins (and presumably functions) being added in a lineage-specific fashion to an ancient, highly conserved functional core, much of which was contributed by the presumptive alpha-proteobacterial symbiont from which the mitochondrial genome was derived.

An early evolutionary origin for the minor spliceosome.

The minor spliceosome is a ribonucleoprotein complex that catalyses the removal of an atypical class of spliceosomal introns (U12-type) from eukaryotic messenger RNAs. It was first identified and characterized in animals, where it was found to contain several unique RNA constituents that share structural similarity with and seem to be functionally analogous to the small nuclear RNAs (snRNAs) contained in the major spliceosome. Subsequently, minor spliceosomal components and U12-type introns have been found in plants but not in fungi. Unlike that of the major spliceosome, which arose early in the eukaryotic lineage, the evolutionary history of the minor spliceosome is unclear because there is evidence of it in so few organisms. Here we report the identification of homologues of minor-spliceosome-specific proteins and snRNAs, and U12-type introns, in distantly related eukaryotic microbes (protists) and in a fungus (Rhizopus oryzae). Cumulatively, our results indicate that the minor spliceosome had an early origin: several of its characteristic constituents are present in representative organisms from all eukaryotic supergroups for which there is any substantial genome sequence information. In addition, our results reveal marked evolutionary conservation of functionally important sequence elements contained within U12-type introns and snRNAs.

Gene discovery in the Acanthamoeba castellanii genome.

Acanthamoeba castellanii is a free-living amoeba found in soil, freshwater, and marine environments and an important predator of bacteria. Acanthamoeba castellanii is also an opportunistic pathogen of clinical interest, responsible for several distinct diseases in humans. In order to provide a genomic platform for the study of this ubiquitous and important protist, we generated a sequence survey of approximately 0.5 x coverage of the genome. The data predict that A. castellanii exhibits a greater biosynthetic capacity than the free-living Dictyostelium discoideum and the parasite Entamoeba histolytica, providing an explanation for the ability of A. castellanii to inhabit a diversity of environments. Alginate lyase may provide access to bacteria within biofilms by breaking down the biofilm matrix, and polyhydroxybutyrate depolymerase may facilitate utilization of the bacterial storage compound polyhydroxybutyrate as a food source. Enzymes for the synthesis and breakdown of cellulose were identified, and they likely participate in encystation and excystation as in D. discoideum. Trehalose-6-phosphate synthase is present, suggesting that trehalose plays a role in stress adaptation. Detection and response to a number of stress conditions is likely accomplished with a large set of signal transduction histidine kinases and a set of putative receptor serine/threonine kinases similar to those found in E. histolytica. Serine, cysteine and metalloproteases were identified, some of which are likely involved in pathogenicity.

Widespread and extensive editing of mitochondrial mRNAS in dinoflagellates.

We report evidence of extensive substitutional editing of mitochondrial mRNAs in the dinoflagellate species Pfiesteria piscicida, Prorocentrum minimum and Crypthecodinium cohnii, based on a comparison of genomic and corresponding cDNA sequences determined for two mitochondrial DNA-encoded genes, cox1 (cytochrome oxidase subunit 1) and cob (apocytochrome b). In the cox1 mRNA, we identify 72 substitutions at 40 sites in 39 codons, whereas in cob mRNA, we infer 86 editing events at 51 sites in 48 codons. Editing, which takes place in distinct clusters, changes approximately 2% of the total sequence, occurs predominantly at first and second positions of codons, and involves mostly (but not exclusively) A-->G (47%), U-->C (23%) and C-->U (17%) substitutions. In all but four of the 158 cases, editing changes the identity of the specified amino acid. At 21 (cox1) and 26 (cob) sites, the same nucleotide change is observed at the same position in at least two of the species investigated. At about one-third of the sites, editing results in an amino acid change that increases similarity between the dinoflagellate Cox1 and Cob sequences and their homologs in other organisms; presumably editing at these sites is of particular functional significance. Overall, about half of the editing events either maintain or increase similarity between the dinoflagellate protein sequences and their non-dinoflagellate homologs, while a further one-third of the alterations are "dinoflagellate-specific" (i.e. they involve a change to an amino acid residue selectively conserved in at least two of the dinoflagellate species at a given position). The nature, pattern and phylogenetic distribution of the inferred edits implies either that more than one type of previously described editing process operates on a given transcript in dinoflagellate mitochondria, or that a mechanistically unique type of mitochondrial mRNA editing has evolved within the dinoflagellate lineage.

Propagule type influences competition between two submersed aquatic macrophytes.

Glasshouse competition experiments with Hydrilla verticillata (L.f.) Royle indicate that plants grown from turions are weaker competitors than those grown from tubers, when compared to the widely distributed macrophyte, Potamogeton pectinatus L. These results support an earlier hypothesis about the importance of propagule size for predicting the outcome of plant competition (Grace 1985; Schaffer and Gadgil 1980). Results of outdoor growth experiments indicate that even though Hydrilla plants from turions are relatively weaker competitors, they are able to grow succesfully in an existing macrophyte bed composed of either, P. pectinatus or P. gramineus. During the early stages of Hydrilla invasion into an area of existing macrophytes, native plants may coexist with Hydrilla. However, once the abundance of Hydrilla tubers in the sediment increases, Hydrilla may displace existing plants.