Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish.

Many eukaryotic genes play essential roles in multiple biological processes in several different tissues. Conditional mutants are needed to analyze genes with such pleiotropic functions. In vertebrates, conditional gene inactivation has only been feasible in the mouse, leaving other model systems to rely on surrogate experimental approaches such as overexpression of dominant negative proteins and antisense-based tools. Here, we have developed a simple and straightforward method to integrate loxP sequences at specific sites in the zebrafish genome using the CRISPR/Cas9 technology and oligonucleotide templates for homology directed repair. We engineered conditional (floxed) mutants of tbx20 and fleer, and demonstrate excision of exons flanked by loxP sites using tamoxifen-inducible CreERT2 recombinase. To demonstrate broad applicability of our method, we also integrated loxP sites into two additional genes, aldh1a2 and tcf21. The ease of this approach will further expand the use of zebrafish to study various aspects of vertebrate biology, especially post-embryonic processes such as regeneration.

Water relations in tree physiology: where to from here?

We look back over 50 years of research into the water relations of trees, with the objective of assessing the maturity of the topic in terms of the idea of a paradigm, put forward by Kuhn in 1962. Our brief review indicates that the physical processes underlying the calculation of transpiration are well understood and accepted, and knowledge of those processes can be applied if information about the leaf area of trees, and stomatal conductance, is available. Considerable progress has been made in understanding the factors governing stomatal responses to environment, with insights into how the hydraulic conducting system of trees determines the maximum aperture of stomata. Knowledge about the maximum stomatal conductance values likely to be reached by different species, and recognition that stomatal responses to increasing atmospheric vapor pressure deficits are in fact responses to water loss from leaves, provides the basis for linking these responses to information about hydraulic conductance through soil­root­stem­branch systems. Improved understanding in these areas is being incorporated into modern models of stomatal conductance and responses to environmental conditions. There have been significant advances in understanding hydraulic pathways, including cavitation and its implications. A few studies suggest that the major resistances to water flux within trees are not in the stem but in the branches. This insight may have implications for productivity: it may be advantageous to select trees with the genetic propensity to produce short branches in stands with open canopies. Studies on the storage of water in stems have provided improved understanding of fluxes from sapwood at different levels. Water stored in the stems of large trees may provide up to 20­30% daily sap flow, but this water is likely to be replaced by inflows at night. In dry conditions transpiration by large trees may be maintained from stored water for up to a week, but flows from storage may be more important in refilling cavitated xylem elements and hence ensuring that the overall hydraulic conductivity of stems is not reduced. Hydraulic redistribution of water in the soil may make a contribution to facilitating root growth in dry soils and modifying resource availability. We conclude that the field of tree water relations is mature, in the sense that the concepts underlying models describing processes and system responses to change are well-tested and accepted and there are few, if any, serious anomalies emerging. Models are essentially formal statements about the way we think systems work. They are always subject to further testing, refinement and improvements. Gaps in knowledge appear within the framework of accepted concepts and mechanisms research is needed to fill those gaps. The models currently available can be used to scale estimates of transpiration from leaf to landscape levels and predict species responses to drought. The focus in tree water relations has shifted to examine the climatic thresholds at which drought, high temperatures and vapor pressure deficits cause mortality. Tree death may be caused by hydraulic collapse following irreversible cavitation or extremely low water potentials, but recent research indicates that the relative sensitivity of stomatal conductance and whole-plant hydraulic conductance plays a major role in determining plant responses to drought.

An ecoregion assessment of projected tree species vulnerabilities in western North America through the 21st century.

Forest ecosystems across western North America will likely see shifts in both tree species dominance and composition over the rest of this century in response to climate change. Our objective in this study was to identify which ecological regions might expect the greatest changes to occur. We used the process-based growth model 3-PG, to provide estimates of tree species responses to changes in environmental conditions and to evaluate the extent that species are resilient to shifts in climate over the rest of this century. We assessed the vulnerability of 20 tree species in western North America using the Canadian global circulation model under three different emission scenarios. We provided detailed projections of species shifts by including soil maps that account for the spatial variation in soil water availability and soil fertility as well as by utilizing annual climate projections of monthly changes in air temperature, precipitation, solar radiation, vapor pressure deficit and frost at a spatial resolution of one km. Projected suitable areas for tree species were compared to their current ranges based on observations at >40 000 field survey plots. Tree species were classified as vulnerable if environmental conditions projected in the future appear outside that of their current distribution ≥70% of the time. We added a migration constraint that limits species dispersal to <200 m yr-1 to provide more realistic projections on species distributions. Based on these combinations of constraints, we predicted the greatest changes in the distribution of dominant tree species to occur within the Northwest Forested Mountains and the highest number of tree species stressed will likely be in the North American Deserts. Projected climatic changes appear especially unfavorable for species in the subalpine zone, where major shifts in composition may lead to the emergence of new forest types.

Progress in Remote Sensing of Photosynthetic Activity over the Amazon Basin.

Although quantifying the massive exchange of carbon that takes place over the Amazon Basin remains a challenge, progress is being made as the remote sensing community moves from using traditional, reflectance-based vegetation indices, such as the Normalized Difference Vegetation Index (NDVI), to the more functional Photochemical Reflectance Index (PRI). This new index, together with satellite-derived estimates of canopy light interception and Sun-Induced Fluorescence (SIF), provide improved estimates of Gross Primary Production (GPP). This paper traces the development of these new approaches, compares the results of their analyses from multiple years of data acquired across the Amazon Basin and suggests further improvements in instrument design, data acquisition and processing. We demonstrated that our estimates of PRI are in generally good agreement with eddy-flux tower measurements of photosynthetic light use efficiency (ε) at four sites in the Amazon Basin: r2 values ranged from 0.37 to 0.51 for northern flux sites and to 0.78 for southern flux sites. This is a significant advance over previous approaches seeking to establish a link between global-scale photosynthetic activity and remotely-sensed data. When combined with measurements of Sun-Induced Fluorescence (SIF), PRI provides realistic estimates of seasonal variation in photosynthesis over the Amazon that relate well to the wet and dry seasons. We anticipate that our findings will steer the development of improved approaches to estimate photosynthetic activity over the tropics.

Internal epitope tagging informed by relative lack of sequence conservation.

Many experimental techniques rely on specific recognition and stringent binding of proteins by antibodies. This can readily be achieved by introducing an epitope tag. We employed an approach that uses a relative lack of evolutionary conservation to inform epitope tag site selection, followed by integration of the tag-coding sequence into the endogenous locus in zebrafish. We demonstrate that an internal epitope tag is accessible for antibody binding, and that tagged proteins retain wild type function.

Predicting large wildfires across western North America by modeling seasonal variation in soil water balance.

A lengthening of the fire season, coupled with higher temperatures, increases the probability of fires throughout much of western North America. Although regional variation in the frequency of fires is well established, attempts to predict the occurrence of fire at a spatial resolution <10 km2 have generally been unsuccessful. We hypothesized that predictions of fires might be improved if depletion of soil water reserves were coupled more directly to maximum leaf area index (LAImax) and stomatal behavior. In an earlier publication, we used LAImax and a process-based forest growth model to derive and map the maximum available soil water storage capacity (ASWmax) of forested lands in western North America at l km resolution. To map large fires, we used data products acquired from NASA's Moderate Resolution Imaging Spectroradiometers (MODIS) over the period 2000-2009. To establish general relationships that incorporate the major biophysical processes that control evaporation and transpiration as well as the flammability of live and dead trees, we constructed a decision tree model (DT). We analyzed seasonal variation in the relative availability of soil water (fASW) for the years 2001, 2004, and 2007, representing respectively, low, moderate, and high rankings of areas burned. For these selected years, the DT predicted where forest fires >1 km occurred and did not occur at ~100,000 randomly located pixels with an average accuracy of 69 %. Extended over the decade, the area predicted burnt varied by as much as 50 %. The DT identified four seasonal combinations, most of which included exhaustion of ASW during the summer as critical; two combinations involving antecedent conditions the previous spring or fall accounted for 86 % of the predicted fires. The approach introduced in this paper can help identify forested areas where management efforts to reduce fire hazards might prove most beneficial.

Satellite observed widespread decline in Mongolian grasslands largely due to overgrazing.

The Mongolian Steppe is one of the largest remaining grassland ecosystems. Recent studies have reported widespread decline of vegetation across the steppe and about 70% of this ecosystem is now considered degraded. Among the scientific community there has been an active debate about whether the observed degradation is related to climate, or over-grazing, or both. Here, we employ a new atmospheric correction and cloud screening algorithm (MAIAC) to investigate trends in satellite observed vegetation phenology. We relate these trends to changes in climate and domestic animal populations. A series of harmonic functions is fitted to Moderate Resolution Imaging Spectroradiometer (MODIS) observed phenological curves to quantify seasonal and inter-annual changes in vegetation. Our results show a widespread decline (of about 12% on average) in MODIS observed normalized difference vegetation index (NDVI) across the country but particularly in the transition zone between grassland and the Gobi desert, where recent decline was as much as 40% below the 2002 mean NDVI. While we found considerable regional differences in the causes of landscape degradation, about 80% of the decline in NDVI could be attributed to increase in livestock. Changes in precipitation were able to explain about 30% of degradation across the country as a whole but up to 50% in areas with denser vegetation cover (P < 0.05). Temperature changes, while significant, played only a minor role (r(2)  = 0.10, P < 0.05). Our results suggest that the cumulative effect of overgrazing is a primary contributor to the degradation of the Mongolian steppe and is at least partially responsible for desertification reported in previous studies.

Evaluating theories of drought-induced vegetation mortality using a multimodel-experiment framework.

SUMMARY: Model-data comparisons of plant physiological processes provide an understanding of mechanisms underlying vegetation responses to climate. We simulated the physiology of a piñon pine-juniper woodland (Pinus edulis-Juniperus monosperma) that experienced mortality during a 5 yr precipitation-reduction experiment, allowing a framework with which to examine our knowledge of drought-induced tree mortality. We used six models designed for scales ranging from individual plants to a global level, all containing state-of-the-art representations of the internal hydraulic and carbohydrate dynamics of woody plants. Despite the large range of model structures, tuning, and parameterization employed, all simulations predicted hydraulic failure and carbon starvation processes co-occurring in dying trees of both species, with the time spent with severe hydraulic failure and carbon starvation, rather than absolute thresholds per se, being a better predictor of impending mortality. Model and empirical data suggest that limited carbon and water exchanges at stomatal, phloem, and below-ground interfaces were associated with mortality of both species. The model-data comparison suggests that the introduction of a mechanistic process into physiology-based models provides equal or improved predictive power over traditional process-model or empirical thresholds. Both biophysical and empirical modeling approaches are useful in understanding processes, particularly when the models fail, because they reveal mechanisms that are likely to underlie mortality. We suggest that for some ecosystems, integration of mechanistic pathogen models into current vegetation models, and evaluation against observations, could result in a breakthrough capability to simulate vegetation dynamics.

Satellite-derived estimates of forest leaf area index in southwest Western Australia are not tightly coupled to interannual variations in rainfall: implications for groundwater decline in a drying climate.

There is increasing concern that widespread forest decline could occur in regions of the world where droughts are predicted to increase in frequency and severity as a result of climate change. The average annual leaf area index (LAI) is an indicator of canopy cover and the difference between the annual maximum and minimum LAI is an indicator of annual leaf turnover. In this study, we analyzed satellite-derived estimates of monthly LAI across forested coastal catchments of southwest Western Australia over a 12 year period (2000-2011) that included the driest year on record for the last 60 years. We observed that over the 12 year study period, the spatial pattern of average annual satellite-derived LAI values was linearly related to mean annual rainfall. However, interannual changes to LAI in response to changes in annual rainfall were far less than expected from the long-term LAI-rainfall trend. This buffered response was investigated using a physiological growth model and attributed to availability of deep soil moisture and/or groundwater storage. The maintenance of high LAIs may be linked to a long-term decline in areal average underground water storage and diminished summer flows, with an emerging trend toward more ephemeral flow regimes.

Predicting tree diversity across the United States as a function of modeled gross primary production.

At the regional and continental scale, ecologists have theorized that spatial variation in biodiversity can be interpreted as a response to differences in climate. To test this theory we assumed that ecological constraints associated with current climatic conditions (2000-2004) might best be correlated with tree richness if expressed through satellite-derived measures of gross primary production (GPP), rather than the more commonly used, but less consistently derived, net primary production. To evaluate current patterns in tree diversity across the contiguous United States we acquired information on tree composition from the USDA Forest Service's Forest Inventory and Analysis program that represented more than 17,4000 survey plots. We selected 2693 cells of 1000 km2 within which a sufficient number of plots were available to estimate tree richness per hectare. Our estimates of forest productivity varied from simple vegetation indices indicative of the fraction of light intercepted by canopies at 16-d intervals, a product from the MODIS (Moderate Resolution Imaging Spectro-radiometer), to 8- and 10-d GPP products derived with minimal climatic data (MODIS) and SPOT-Vegetation (Systeme Pour l'Observation de la Terre), to 3-PGS (Physiological Principles Predicting Growth with Satellites), which requires both climate and soil data. Across the contiguous United States, modeled predictions of gross productivity accounted for between 51% and 77% of the recorded spatial variation in tree diversity, which ranged from 2 to 67 species per hectare. When the analyses were concentrated within nine broadly defined ecoregions, predictive relations largely disappeared. Only 3-PGS predictions fit a theorized unimodal function by being able to distinguish highly productive forests in the Pacific Northwest that support lower than expected tree diversity. Other models predicted a continuous steep rise in tree diversity with increasing productivity, and did so with generally better or nearly equal precision with fewer data requirements.

Physiological girdling of pine trees via phloem chilling: proof of concept.

Quantifying below-ground carbon (C) allocation is particularly difficult as methods usually disturb the root-mycorrhizal-soil continuum. We reduced C allocation below ground of loblolly pine trees by: (1) physically girdling trees and (2) physiologically girdling pine trees by chilling the phloem. Chilling reduced cambium temperatures by approximately 18 degrees C. Both methods rapidly reduced soil CO2 efflux, and after approximately 10 days decreased net photosynthesis (P(n)), the latter indicating feedback inhibition. Chilling decreased soil-soluble C, indicating that decreased soil CO2 efflux may have been mediated by a decrease in root C exudation that was rapidly respired by microbes. These effects were only observed in late summer/early autumn when above-ground growth was minimal, and not in the spring when above-ground growth was rapid. All of the effects were rapidly reversed when chilling was ceased. In fertilized plots, both chilling and physical girdling methods reduced soil CO2 efflux by approximately 8%. Physical girdling reduced soil CO2 efflux by 26% in non-fertilized plots. This work demonstrates that phloem chilling provides a non-destructive alternative to reducing the movement of recent photosynthate below the point of chilling to estimate C allocation below ground on large trees.

Gas-phase rate coefficients for the reactions of nitrate radicals with (Z)-pent-2-ene, (E)-pent-2-ene, (Z)-hex-2-ene, (E)-hex-2-ene, (Z)-hex-3-ene, (E)-hex-3-ene and (E)-3-methylpent-2-ene at room temperature.

Rate coefficients for reactions of nitrate radicals (NO3) with (Z)-pent-2-ene, (E)-pent-2-ene, (Z)-hex-2-ene, (E)-hex-2-ene, (Z)-hex-3-ene, (E)-hex-3-ene and (E)-3-methylpent-2-ene were determined to be (6.55 +/- 0.78)x 10(-13) cm3 molecule(-1) s(-1), (3.78 +/- 0.45)x 10(-13) cm3 molecule(-1) s(-1), (5.30 +/- 0.73)x 10(-13) cm(3) molecule(-1) s(-1), (3.83 +/- 0.47)x 10(-13) cm(3) molecule(-1) s(-1), (4.37 +/- 0.49)x 10(-13) cm(3) molecule(-1) s(-1), (3.61 +/- 0.40)x 10(-13) cm3 molecule(-1) s(-1) and (8.9 +/- 1.5)x 10(-12) cm3 molecule(-1) s(-1), respectively. We performed kinetic experiments at room temperature and atmospheric pressure using a relative-rate technique with GC-FID analysis. The experimental results demonstrate a surprisingly large cis-trans(Z-E) effect, particularly in the case of the pent-2-enes, where the ratio of rate coefficients is ca. 1.7. Rate coefficients are discussed in terms of electronic and steric influences, and our results give some insight into the effects of chain length and position of the double bond on the reaction of NO3 with unsaturated hydrocarbons. Atmospheric lifetimes were calculated with respect to important oxidants in the troposphere for the alkenes studied, and NO3-initiated oxidation is found to be the dominant degradation route for (Z)-pent-2-ene, (Z)-hex-3-ene and (E)-3-methylpent-2-ene.

Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor.

We determined the crystal structure of a bifunctional group I intron splicing factor and homing endonuclease, termed the I-AniI maturase, in complex with its DNA target at 2.6 A resolution. The structure demonstrates the remarkable structural conservation of the beta-sheet DNA-binding motif between highly divergent enzyme subfamilies. DNA recognition by I-AniI was further studied using nucleoside deletion and DMS modification interference analyses. Correlation of these results with the crystal structure provides information on the relative importance of individual nucleotide contacts for DNA recognition. Alignment and modeling of two homologous maturases reveals conserved basic surface residues, distant from the DNA-binding surface, that might be involved in RNA binding. A point mutation that introduces a single negative charge in this region uncouples the maturase and endonuclease functions of the protein, inhibiting RNA binding and splicing while maintaining DNA binding and cleavage.

In vitro analysis of the relationship between endonuclease and maturase activities in the bi-functional group I intron-encoded protein, I-AniI.

The AnCOB group I intron from Aspergillus nidulans encodes a homing DNA endonuclease called I-AniI which also functions as a maturase, assisting in AnCOB intron RNA splicing. In this investigation we biochemically characterized the endonuclease activity of I-AniI in vitro and utilized competition assays to probe the relationship between the RNA- and DNA-binding sites. Despite functioning as an RNA maturase, I-AniI still retains several characteristic properties of homing endonucleases including relaxed substrate specificity, DNA cleavage product retention and instability in the reaction buffer, which suggest that the protein has not undergone dramatic structural adaptations to function as an RNA-binding protein. Nitrocellulose filter binding and kinetic burst assays showed that both nucleic acids bind I-AniI with the same 1 : 1 stoichiometry. Furthermore, in vitro competition activity assays revealed that the RNA substrate, when prebound to I-AniI, stoichiometrically inhibits DNA cleavage activity, yet in reciprocal experiments, saturating amounts of prebound DNA substrate fails to inhibit RNA splicing activity. The data suggest therefore that both nucleic acids do not bind the same single binding site, rather that I-AniI appears to contain two binding sites.

Lessons learned while extending physiological principles from growth chambers to satellite studies.

Over the last three decades, physiological principles established in laboratory studies have been applied to systems at progressively larger scales and are now firmly merged into the fields of ecology, ecosystem modeling, forest protection, and global change research. To expand the vision of any field requires that scientists from different disciplines build a bridge across the chasm that normally exists between the knowledge bases and perspectives of different fields. Bridges are built most quickly when representatives of different disciplines see the possibility of mutual advantage in collaboration and seek to quickly demonstrate that potential. Usually, however, the process is laborious because approaches and techniques must be modified to address problems at a different level of integration. Successful bridge builders have, almost without exception, established credibility in their own field and have then identified a kindred spirit with similar credentials in another. They usually establish a pilot study that involves apprentices as well as established scientists. If the approach is successful, the younger members of the team often take the lead in further advancements. Managers of large centralized programs should foster interdisciplinary exchange, particularly at times when advancement in one field languishes. To expand collaboration, it is often necessary for scientists to seek common properties that simplify relations across a wide range of biological and physical conditions. This integrative perspective is essential and is fostered by participating in cross-disciplinary workshops and conferences and by reading outside one's field.

Effects of branch length on carbon isotope discrimination in Pinus radiata.

Gas exchange was measured on a pruned Pinus radiata D. Don hedge and on a long-branch P. radiata tree near Hamilton, New Zealand, in spring 1993 when soil water content was close to field capacity. Foliage at the end of long branches (9.0 m) showed a marked drop in net photosynthetic rate and stomatal conductance as the saturation deficit increased, whereas foliage on short branches (0.5 m) showed little change. Mean foliage delta(13)C was -30.1 per thousand for short branches and -26.3 per thousand for long branches. Foliage delta(13)C was correlated with branch length in two genetically improved P. radiata seedlots at four stocking densities. The multinodal seedlot had shorter branches and more (13)C-depleted foliage compared with branches and foliage from the long internode seedlot. There was a strong effect of stocking density on carbon isotope composition in both seedlots. We conclude that branch morphology affects foliage gas exchange properties and foliage carbon isotope composition.

Woody tissue maintenance respiration of four conifers in contrasting climates.

We estimate maintenance respiration for boles of four temperate conifers (ponderosa pine, western hemlock, red pine, and slash pine) from CO2 efflux measurements in autumn, when construction respiration is low or negligible. Maintenance respiration of stems was linearly related to sapwood volume for all species; at 10°C, respiration per unit sapwood volume ranged from 4.8 to 8.3 µmol CO2 m-3 s-1. For all sites combined, respiration increased exponentially with temperature (Q 10 =1.7, r 2=0.78). We estimate that maintenance respiration of aboveground woody tissues of these conifers consumes 52-162 g C m-2 y-1, or 5-13% of net daytime carbon assimilation annually. The fraction of annual net daytime carbon fixation used for stem maintenance respiration increased linearly with the average annual temperature of the site.