Impact of different levels of cinnamyl alcohol dehydrogenase down-regulation on lignins of transgenic tobacco plants

The effects of cinnamyl alcohol dehydrogenase (CAD, EC.1.1.1.195) down-regulation on lignin profiles of plants were analysed in four selected transgenic lines of tobacco (Nicotiana tabacum L. cv. Samsun) exhibiting different levels of CAD activity (8–56% of the control). A significant decrease in thioacidolysis yields (i.e. yield of β-O-4 linked monomers) and in the ratio of syringyl to guaiacyl monomers (S/G) was observed for three transgenic lines and the most drastic reduction (up to 50%) was correlated with the lowest level of CAD activity. Higher lignin extractability by mild alkali treatment was confirmed, and, in addition to a tenfold increase in C₆-C₁ aldehydes, coniferyl aldehyde was detected by high-performance liquid chromatography in the alkali extracts from the xylem of transgenic plants. In-situ polymerisation of cinnamyl aldehydes in stem sections of untransformed tobacco gave a xylem cell wall coloration strikingly similar to the reddish-brown coloration of the xylem of antisense CAD-down-regulated plants. Overall, these data provide new arguments for the involvement of polymerised cinnamyl aldehydes in the formation of the red-coloured xylem of CAD-down-regulated plants. Received: 24 January 1997 / Accepted: 14 May 1997     

Characterization of the Golgi Complex Cleared of Proteins in Transit and Examination of Calcium Uptake Activities

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were >99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that >90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.     

Side-chain cleavage of cholesterol esters by human cytochrome P-450scc

In order to define the substrate binding site of human cytochrome P-450_scc in the vicinity of the 3β-hydroxyl group of cholesterol, we have tested the ability of the cytochrome to cleave the side chain of a range of cholesterol esters and cholesterol methyl ether. Using a Tween-20 detergent reconstituted system we found that cholesterol sulphate could undergo side-chain cleavage with the same turnover number (k_cat) as that for cholesterol, but with a higher K_m. Cholesterol methyl ether underwent side-chain cleavage to pregnenolone methyl ether with k_cat and K_m values 30% of those for cholesterol. Cholesterol fatty acid esters with acyl chain lengths of up to four carbons were able to undergo side-chain cleavage with K_m values similar to those for cholesterol, but k_cat values only 12–23% of those for cholesterol. Turnover numbers decreased as the acyl group length increased beyond four carbons, although some activity was still detected with cholesterol palmitate as substrate. Analysis of bovine cytochrome P-450_scc revealed that it could also cleave the side chain of acyl and sulphate esters of cholesterol. This study indicates that the substrate binding site of cytochrome P-450_scc in the vicinity of the 3β-hydroxyl group is larger than previously believed.     

Molecular Characterization of the Clusters of Genes Encoding the Botulinum Neurotoxin Complex in Clostridium botulinum (Clostridium argentinense) Type G and Nonproteolytic Clostridium botulinum Type B

The cluster of genes encoding components of the progenitor botulinum neurotoxin complex has been mapped and cloned in Clostridium botulinum type G strain ATCC 27322. Determination of the nucleotide sequence of the region has revealed open reading frames encoding nontoxic components of the complex, upstream of the gene encoding BoNT/G (botG). The arrangement of these genes differs from that in strains of other antigenic toxin types. Immediately upstream of botG lies a gene encoding a protein of 1198 amino acids, which shows homology with the nontoxic-nonhemagglutinin (NTNH) component of the progenitor complex. Further upstream there are genes encoding proteins with homology to hemagglutinin components (HA-17, HA-70) and a putative positive regulator of gene expression (P-21). Sequence comparison has shown that BoNT/G has highest homology with BoNT/B. The sequence of the BoNT-cluster of genes in non-proteolytic C. botulinum type B strain Eklund 17B has been extended to include the complete NTNH and HA-17, and partial HA-70 gene sequences. Comparison of NTNH/G with other NTNHs reveals that it shows highest homology with NTNH/B consistent with the genealogical affinity shown between BoNT/G and BoNT/B genes. Received: 28 January 1997 / Accepted: 24 March 1997     

Development of real-time PCR (TaqMan) assays for the detection and quantification of Botrytis cinerea in planta.

Real-time PCR assays based on TaqMan chemistry have been developed for the detection and quantification of Botrytis cinerea, suitable for a wide range of different host plant species. Assays were designed to the beta-tubulin gene, the intergenic spacer (IGS) region of the nuclear ribosomal DNA and also to a previously published, species-specific sequence characterised amplified region (SCAR) marker; the assays were compared to a published method based on SYBR Green I technology. The assays designed to the IGS region and SCAR marker proved to be highly specific for B. cinerea but assays designed to the beta-tubulin gene and the previously published assay designed to the cutinase-A gene both cross-react with B. fabae. The assay designed to the IGS region was the most sensitive and was able to reliably detect and quantify as little as 20 fg of B. cinerea DNA. The method incorporates the detection of a gene from the plant host to compensate for variations in extraction efficiency and size of sample tested. The assays designed were used to follow the progression of infection of B. cinerea in plant material inoculated with spores to the point of symptom induction. They should be ideally suited to investigating infection processes in-planta and could be used to investigate aspects of infection/plant pathogenesis, by B. cinerea and are particularly suited to the detection and quantification of the pathogen prior to the development of symptoms.     

Land cover change and carbon emissions over 100 years in an African biodiversity hotspot

Agricultural expansion has resulted in both land use and land cover change (LULCC) across the tropics. However, the spatial and temporal patterns of such change and their resulting impacts are poorly understood, particularly for the presatellite era. Here, we quantify the LULCC history across the 33.9 million ha watershed of Tanzania's Eastern Arc Mountains, using geo‐referenced and digitized historical land cover maps (dated 1908, 1923, 1949 and 2000). Our time series from this biodiversity hotspot shows that forest and savanna area both declined, by 74% (2.8 million ha) and 10% (2.9 million ha), respectively, between 1908 and 2000. This vegetation was replaced by a fivefold increase in cropland, from 1.2 million ha to 6.7 million ha. This LULCC implies a committed release of 0.9 Pg C (95% CI: 0.4–1.5) across the watershed for the same period, equivalent to 0.3 Mg C ha^?1 yr^?1. This is at least threefold higher than previous estimates from global models for the same study area. We then used the LULCC data from before and after protected area creation, as well as from areas where no protection was established, to analyse the effectiveness of legal protection on land cover change despite the underlying spatial variation in protected areas. We found that, between 1949 and 2000, forest expanded within legally protected areas, resulting in carbon uptake of 4.8 (3.8–5.7) Mg C ha^?1, compared to a committed loss of 11.9 (7.2–16.6) Mg C ha^?1 within areas lacking such protection. Furthermore, for nine protected areas where LULCC data are available prior to and following establishment, we show that protection reduces deforestation rates by 150% relative to unprotected portions of the watershed. Our results highlight that considerable LULCC occurred prior to the satellite era, thus other data sources are required to better understand long‐term land cover trends in the tropics.     

Predation of artificial nests by introduced rhesus macaques (<Emphasis Type="Italic">Macaca mulatta</Emphasis>) in Florida,USA

Native throughout Asia, rhesus macaques are believed to have the widest native range of any non-human primate and are capable of adapting to an extensive diversity of habitats. Rhesus macaques have caused environmental degradation in introduced habitats, including decreasing bird populations through nest predation. In the 1930s, rhesus macaques were intentionally introduced into what is today Silver Springs State Park (SSSP), central Florida, in an effort to increase tourism. Our objective was to determine whether introduced rhesus macaques in SSSP would consume eggs presented in artificial nests. We used camera traps adjacent to 100 open-cupped artificial bird nests baited with quail eggs near the Silver River. Nests were placed in shrubs and left in the field site for 12 days, representative of the incubation period of native passerine species. Twenty-one nests were depredated by rhesus macaques, nine by nest predators other than macaques, and five nests by an unidentified predator. Nests were more likely to be depredated by macaques when located in areas of high macaque relative abundance. This study suggests introduced rhesus macaques may influence nest predation rates of native bird species in natural areas.     

An Evolutionary View of the Arms Race between Protein Kinase R and Large DNA Viruses

To establish productive infections, viruses must counteract numerous cellular defenses that are poised to recognize viruses as nonself and to activate antiviral pathways. The opposing goals of host and viral factors lead to evolutionary arms races that can be illuminated by evolutionary and computational methods and tested in experimental models. Here we illustrate how this perspective has been contributing to our understanding of the interactions of the protein kinase R pathway with large DNA viruses.     

Intrapopulation genomics in a model mutualist: Population structure and candidate symbiosis genes under selection in Medicago truncatula

Bottom‐up evolutionary approaches, including geographically explicit population genomic analyses, have the power to reveal the mechanistic basis of adaptation. Here, we conduct a population genomic analysis in the model legume, Medicago truncatula, to characterize population genetic structure and identify symbiosis‐related genes showing evidence of spatially variable selection. Using RAD‐seq, we generated over 26,000 SNPs from 191 accessions from within three regions of the native range in Europe. Results from STRUCTURE analysis identify five distinct genetic clusters with divisions that separate east and west regions in the Mediterranean basin. Much of the genetic variation is maintained within sampling sites, and there is evidence for isolation by distance. Extensive linkage disequilibrium was identified, particularly within populations. We conducted genetic outlier analysis with F_ST‐based genome scans and a Bayesian modeling approach (PCAdapt). There were 70 core outlier loci shared between these distinct methods with one clear candidate symbiosis related gene, DMI1. This work sets that stage for functional experiments to determine the important phenotypes that selection has acted upon and complementary efforts in rhizobium populations.