A Global Analysis of Deforestation in Moist Tropical Forest Protected Areas.

Protected areas (PAs) have been established to conserve tropical forests, but their effectiveness at reducing deforestation is uncertain. To explore this issue, we combined high resolution data of global forest loss over the period 2000-2012 with data on PAs. For each PA we quantified forest loss within the PA, in buffer zones 1, 5, 10 and 15 km outside the PA boundary as well as a 1 km buffer within the PA boundary. We analysed 3376 tropical and subtropical moist forest PAs in 56 countries over 4 continents. We found that 73% of PAs experienced substantial deforestation pressure, with >0.1% a(-1) forest loss in the outer 1 km buffer. Forest loss within PAs was greatest in Asia (0.25% a(-1)) compared to Africa (0.1% a(-1)), the Neotropics (0.1% a(-1)) and Australasia (Australia and Papua New Guinea; 0.03% a(-1)). We defined performance (P) of a PA as the ratio of forest loss in the inner 1 km buffer compared to the loss that would have occurred in the absence of the PA, calculated as the loss in the outer 1 km buffer corrected for any difference in deforestation pressure between the two buffers. To remove the potential bias due to terrain, we analysed a subset of PAs (n = 1804) where slope and elevation in inner and outer 1 km buffers were similar (within 1° and 100 m, respectively). We found 41% of PAs in this subset reduced forest loss in the inner buffer by at least 25% compared to the expected inner buffer forest loss (P<0.75). Median performance (P) of subset reserves was 0.87, meaning a reduction in forest loss within the PA of 13%. We found PAs were most effective in Australasia (P = 0.16), moderately successful in the Neotropics (P = 0.72) and Africa (p = 0.83), but ineffective in Asia (P = 1). We found many countries have PAs that give little or no protection to forest loss, particularly in parts of Asia, west Africa and central America. Across the tropics, the median effectiveness of PAs at the national level improved with gross domestic product per capita. Whilst tropical and subtropical moist forest PAs do reduce forest loss, widely varying performance suggests substantial opportunities for improved protection, particularly in Asia.

Death from drought in tropical forests is triggered by hydraulics not carbon starvation.

Drought threatens tropical rainforests over seasonal to decadal timescales, but the drivers of tree mortality following drought remain poorly understood. It has been suggested that reduced availability of non-structural carbohydrates (NSC) critically increases mortality risk through insufficient carbon supply to metabolism ('carbon starvation'). However, little is known about how NSC stores are affected by drought, especially over the long term, and whether they are more important than hydraulic processes in determining drought-induced mortality. Using data from the world's longest-running experimental drought study in tropical rainforest (in the Brazilian Amazon), we test whether carbon starvation or deterioration of the water-conducting pathways from soil to leaf trigger tree mortality. Biomass loss from mortality in the experimentally droughted forest increased substantially after >10 years of reduced soil moisture availability. The mortality signal was dominated by the death of large trees, which were at a much greater risk of hydraulic deterioration than smaller trees. However, we find no evidence that the droughted trees suffered carbon starvation, as their NSC concentrations were similar to those of non-droughted trees, and growth rates did not decline in either living or dying trees. Our results indicate that hydraulics, rather than carbon starvation, triggers tree death from drought in tropical rainforest.

Long-term decline of the Amazon carbon sink.

Atmospheric carbon dioxide records indicate that the land surface has acted as a strong global carbon sink over recent decades, with a substantial fraction of this sink probably located in the tropics, particularly in the Amazon. Nevertheless, it is unclear how the terrestrial carbon sink will evolve as climate and atmospheric composition continue to change. Here we analyse the historical evolution of the biomass dynamics of the Amazon rainforest over three decades using a distributed network of 321 plots. While this analysis confirms that Amazon forests have acted as a long-term net biomass sink, we find a long-term decreasing trend of carbon accumulation. Rates of net increase in above-ground biomass declined by one-third during the past decade compared to the 1990s. This is a consequence of growth rate increases levelling off recently, while biomass mortality persistently increased throughout, leading to a shortening of carbon residence times. Potential drivers for the mortality increase include greater climate variability, and feedbacks of faster growth on mortality, resulting in shortened tree longevity. The observed decline of the Amazon sink diverges markedly from the recent increase in terrestrial carbon uptake at the global scale, and is contrary to expectations based on models.

An evidence-based analysis of epidemiologic associations between lymphatic and hematopoietic cancers and occupational exposure to gasoline.

The presence of benzene in motor gasoline has been a health concern for potential increased risk of acute myelogenous leukemia and perhaps other lymphatic/hematopoietic cancers for approximately 40 years. Because of the widespread and increasing use of gasoline by consumers and the high exposure potential of occupational cohorts, a thorough understanding of this issue is important. The current study utilizes an evidence-based approach to examine whether or not the available epidemiologic studies demonstrate a strong and consistent association between occupational exposure to gasoline and lymphatic/hematopoietic cancers. Among 67 epidemiologic studies initially identified, 54 were ranked according to specific criteria relating to the relevance and robustness of each study for answering the research question. The 30 highest-ranked studies were sorted into three tiers of evidence and were analyzed for strength, specificity, consistency, temporality, dose-response trends and coherence. Meta statistics were also calculated for each general and specific lymphatic/hematopoietic cancer category with adequate data. The evidence-based analysis did not confirm any strong and consistent association between occupational exposure to gasoline and lymphatic/hematopoietic cancers based on the epidemiologic studies available to date. These epidemiologic findings, combined with the evidence showing relatively low occupational benzene vapor exposures associated with gasoline formulations during the last three decades, suggest that current motor gasoline formulations are not associated with increased lymphatic/hematopoietic cancer risks related to benzene.

A high-density quantitative nuclease protection microarray platform for high throughput analysis of gene expression.

The quantitative nuclease protection assay (qNPA) is a very simple and highly sensitive method for measuring mRNA transcripts, can be used on a variety of sample types, and is amenable to high-throughput sample processing. We have combined the power of the qNPA assay with the density of a DNA microarray to create a qNPA Microarray platform. This platform is compatible with common laboratory equipment: it uses fluorescence-based detection, can be analyzed with common microarray scanners, and is in an SBS footprint with 96-well layout for high-throughput applications. Here, we demonstrate the characteristics of a qNPA Microarray slide that contains up to 1700 gene elements per well. We show that the new platform can reliably detect transcripts at levels as low as 10fM with median CVs below 12%. On a standardized set of samples, the qNPA Microarray detected the same trends in gene expression as the original qNPA technology, real time qPCR, and Affymetrix GeneChip DNA Microarrays. Given its ease of use, compatibility with multiple sample types, high-throughput capabilities, and its integration with standard laboratory equipment, the qNPA Microarray is a powerful new platform for gene expression research.

Shifts in plant respiration and carbon use efficiency at a large-scale drought experiment in the eastern Amazon.

*The effects of drought on the Amazon rainforest are potentially large but remain poorly understood. Here, carbon (C) cycling after 5 yr of a large-scale through-fall exclusion (TFE) experiment excluding about 50% of incident rainfall from an eastern Amazon rainforest was compared with a nearby control plot. *Principal C stocks and fluxes were intensively measured in 2005. Additional minor components were either quantified in later site measurements or derived from the available literature. *Total ecosystem respiration (R(eco)) and total plant C expenditure (PCE, the sum of net primary productivity (NPP) and autotrophic respiration (R(auto))), were elevated on the TFE plot relative to the control. The increase in PCE and R(eco) was mainly caused by a rise in R(auto) from foliage and roots. Heterotrophic respiration did not differ substantially between plots. NPP was 2.4 +/- 1.4 t C ha(-1) yr(-1) lower on the TFE than the control. Ecosystem carbon use efficiency, the proportion of PCE invested in NPP, was lower in the TFE plot (0.24 +/- 0.04) than in the control (0.32 +/- 0.04). *Drought caused by the TFE treatment appeared to drive fundamental shifts in ecosystem C cycling with potentially important consequences for long-term forest C storage.

Nuclear DNA content of the whitefly Bemisia tabaci (Aleyrodidae: Hemiptera) estimated by flow cytometry.

The nuclear DNA content of the whitefly Bemisia tabaci (Gennnadius) was estimated using flow cytometry. Male and female nuclei were stained with propidium iodide and their DNA content was estimated using chicken red blood cells and Arabidopsis thaliana L. (Brassicaceae) as external standards. The estimated nuclear DNA content of male and female B. tabaci was 1.04 and 2.06 pg, respectively. These results corroborated previous reports based on chromosome counting, which showed that B. tabaci males are haploid and females are diploid. Conversion between DNA content and genome size (1 pg DNA=980 Mbp) indicate that the haploid genome size of B. tabaci is 1020 Mbp, which is approximately five times the size of the genome of the fruitfly Drosophila melanogaster Meigen. These results provide an important baseline that will facilitate genomics-based research for the B. tabaci complex.

Methods for the exclusion of circoviruses and gammaherpesviruses from pigs.

The use of porcine tissues is being developed as a means to alleviate the shortage of allogeneic tissues and organs available for transplantation. To reduce the possibility of a microorganism of pigs being inadvertently transferred to the recipient of the xenograft, recommendations have been published on the microbiological specifications for organ source pigs. The porcine circoviruses (PCV1 and PCV2) and porcine lymphotropic herpesviruses (PLHV1 and PLHV2) are two infectious agents of pigs which are considered to be of significance for the microbiological safety of xenotransplantation. To ensure the exclusion of these microorganisms from animals destined for use under clinical conditions, reliable breeding methodologies are required. We investigated the efficiency of established derivation procedures for the removal of PCV and PLHV. In comparison with conventionally reared pigs, caesarian and barrier derived animals showed a markedly reduced prevalence of PCVs and PLHVs. Our results indicate that the derivation of animals free of both of these microorganisms is achievable and will enhance the microbiological safety of xenotransplantation.

Transmissible spongiform encephalopathies and tissue cell culture.

The discovery of prion proteins and the diseases which are associated with them still present scientists and clinicians with a number of problems. There are clearly risks with the use of living cells and materials of animal origin to produce therapeutic compounds with respect to the transmission of prion protein. However the medical benefit many of these compounds has to be weighed against this. It is clear a number of groups are continuing to unravel the highly complex relationships of prion biology and pathology and it is only when this is clearly established that the community can decide on these issues. Until this time the scientific community must rely on the best research available and provide guidance from this.

Modulation of abscisic acid signal transduction and biosynthesis by an Sm-like protein in Arabidopsis.

The phytohormone abscisic acid (ABA) regulates plant growth and development as well as stress tolerance. The Arabidopsis sad1 (supersensitive to ABA and drought) mutation increases plant sensitivity to drought stress and ABA in seed germination, root growth, and the expression of some stress-responsive genes. sad1 plants are also defective in the positive feedback regulation of ABA biosynthesis genes by ABA and are impaired in drought stress induction of ABA biosynthesis. SAD1 encodes a polypeptide similar to multifunctional Sm-like snRNP proteins that are required for mRNA splicing, export, and degradation. These results suggest a critical role for mRNA metabolism in the control of ABA signaling as well as in the regulation of ABA homeostasis.

Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba.

One of the most important functions of the plant hormone abscisic acid (ABA) is to induce stomatal closure by reducing the turgor of guard cells under water deficit. Under environmental stresses, hydrogen peroxide (H(2)O(2)), an active oxygen species, is widely generated in many biological systems. Here, using an epidermal strip bioassay and laser-scanning confocal microscopy, we provide evidence that H(2)O(2) may function as an intermediate in ABA signaling in Vicia faba guard cells. H(2)O(2) inhibited induced closure of stomata, and this effect was reversed by ascorbic acid at concentrations lower than 10(-5) M. Further, ABA-induced stomatal closure also was abolished partly by addition of exogenous catalase (CAT) and diphenylene iodonium (DPI), which are an H(2)O(2) scavenger and an NADPH oxidase inhibitor, respectively. Time course experiments of single-cell assays based on the fluorescent probe dichlorofluorescein showed that the generation of H(2)O(2) was dependent on ABA concentration and an increase in the fluorescence intensity of the chloroplast occurred significantly earlier than within the other regions of guard cells. The ABA-induced change in fluorescence intensity in guard cells was abolished by the application of CAT and DPI. In addition, ABA microinjected into guard cells markedly induced H(2)O(2) production, which preceded stomatal closure. These effects were abolished by CAT or DPI micro-injection. Our results suggest that guard cells treated with ABA may close the stomata via a pathway with H(2)O(2) production involved, and H(2)O(2) may be an intermediate in ABA signaling.

Microarray-based analysis of gene expression in very large gene families: the cytochrome P450 gene superfamily of Arabidopsis thaliana.

Cytochrome P450 (P450s) are heme-thiolate protein products of a very large gene superfamily, present in all kingdoms and involved in a variety of metabolic reactions. P450s are classified according to the degree of amino acid sequence identity, with P450s of the same family defined as having >40% identity, and P450s of the same subfamily having >55% identity. Currently, 273 P450 genes distributed over 45 families have been identified in Arabidopsis, and its genome is estimated to contain as many as 286. Genome-wide DNA microarrays make it possible to broadly correlate P450 gene activity with alterations in physiological or developmental states. A potential problem with microarray research is that sequence similarity between and within these families of closely related genes may lead to cross-hybridization. We designed experiments to systematically evaluate the specificity of P450 microarrays, and showed that conditions could be optimized to provide a very high degree of hybridization specificity. Under these conditions, and employing a 20% intensity value of maximum hybridization intensity as a cut-off, labeled P450 genes exhibited essentially no cross-hybridization between families and within subfamilies. We also compared the gene transcription levels of microarray probes derived from EST clones and from genomic DNA sequences for which ESTs were not available, using cDNA produced from RNA from various Arabidopsis tissue as the target. Many of the P450 genes displayed tissue-specific expression, leading to hypotheses as to the function of individual genes and their regulation. We also observed that several of the genomic sequences reported high levels of expression, highlighting the limitations of expression analysis based on ESTs alone.

Gene expression profiles during the initial phase of salt stress in rice.

Transcript regulation in response to high salinity was investigated for salt-tolerant rice (var Pokkali) with microarrays including 1728 cDNAs from libraries of salt-stressed roots. NaCl at 150 mM reduced photosynthesis to one tenth of the prestress value within minutes. Hybridizations of RNA to microarray slides probed for changes in transcripts from 15 min to 1 week after salt shock. Beginning 15 min after the shock, Pokkali showed upregulation of transcripts. Approximately 10% of the transcripts in Pokkali were significantly upregulated or downregulated within 1 hr of salt stress. The initial differences between control and stressed plants continued for hours but became less pronounced as the plants adapted over time. The interpretation of an adaptive process was supported by the similar analysis of salinity-sensitive rice (var IR29), in which the immediate response exhibited by Pokkali was delayed and later resulted in downregulation of transcription and death. The upregulated functions observed with Pokkali at different time points during stress adaptation changed over time. Increased protein synthesis and protein turnover were observed at early time points, followed by the induction of known stress-responsive transcripts within hours, and the induction of transcripts for defense-related functions later. After 1 week, the nature of upregulated transcripts (e.g., aquaporins) indicated recovery.

Microarray-based survey of repetitive genomic sequences in Vicia spp.

A modified DNA microarray-based technique was devised for preliminary screening of short fragment genomic DNA libraries from three Vicia species (V. melanops, V. narbonensis, and V. sativa) to isolate representative highly abundant DNA sequences that show different distribution patterns among related legume species. The microarrays were sequentially hybridized with labeled genomic DNAs of thirteen Vicia and seven other Fabaceae species and scored for hybridization signals of individual clones. The clones were then assigned to one of the following groups characterized by hybridization to: (1) all tested species, (2) most of the Vicia and Pisum species, (3) only a few Vicia species, and (4) preferentially a single Vicia species. Several clones from each group, 65 in total, were sequenced. All Group I clones were identified as rDNA genes or fragments of chloroplast genome, whereas the majority of Group II clones showed significant homologies to retroelement sequences. Clones in Groups III and IV contained novel dispersed repeats with copy numbers 10(2)-10(6)/1C and two genus-specific tandem repeats. One of these belongs to the VicTR-B repeat family, and the other clone (S12) contains an amplified portion of the rDNA intergenic spacer. In situ hybridization using V. sativa metaphase chromosomes revealed the presence of the S12 sequences not only within rDNA genes, but also at several additional loci. The newly identified repeats, as well as the retroelement-like sequences, were characterized with respect to their abundance within individual genomes. Correlations between the repeat distributions and the current taxonomic classification of these species are discussed.

CYP83B1, a cytochrome P450 at the metabolic branch point in auxin and indole glucosinolate biosynthesis in Arabidopsis.

Auxins are growth regulators involved in virtually all aspects of plant development. However, little is known about how plants synthesize these essential compounds. We propose that the level of indole-3-acetic acid is regulated by the flux of indole-3-acetaldoxime through a cytochrome P450, CYP83B1, to the glucosinolate pathway. A T-DNA insertion in the CYP83B1 gene leads to plants with a phenotype that suggests severe auxin overproduction, whereas CYP83B1 overexpression leads to loss of apical dominance typical of auxin deficit. CYP83B1 N-hydroxylates indole-3-acetaldoxime to the corresponding aci-nitro compound, 1-aci-nitro-2-indolyl-ethane, with a K(m) of 3 microM and a turnover number of 53 min(-1). The aci-nitro compound formed reacts non-enzymatically with thiol compounds to produce an N-alkyl-thiohydroximate adduct, the committed precursor of glucosinolates. Thus, indole-3-acetaldoxime is the metabolic branch point between the primary auxin indole-3-acetic acid and indole glucosinolate biosynthesis in Arabidopsis.

Analysis of nuclear DNA content and ploidy in higher plants.

This is the first of a series of units discussing the application of cytometry to plant material. Techniques commonly used for mammalian nuclei evaluation need considerable modification to be successful with plant material. David Galbraith and his colleagues bring together many years of knowledge in plant cytometry. Their unit provides detailed protocols on measuring DNA content, ploidy, and cell cycle status of plant tissue using both conventional laser based instruments as well as arc lamp cytometers. This unit provides an excellent starting point for those interested in doing cytometry with plants.