Assessing vulnerability to embolism and hydraulic safety margins in reed-like Restionaceae.

The African Restionaceae (Poales), the dominant graminoid layer in the megadiverse Cape Floristic Region of South Africa, are distributed across a wide range of moisture availability, yet currently there is very little known about the underlying hydraulics of this group. We tested two methods for measuring culm vulnerability to embolism, the optical and pneumatic methods, in three species of Cannomois ranging in habitat from semi-riparian (Cannomois virgata) to dryland (Cannomois parviflora and C. congesta). Estimates of culm xylem vulnerability were coupled with measures of turgor loss point (ΨTLP) and minimum field water potential (ΨMD) to assess hydraulic safety margins. The optical and pneumatic methods produced similar estimates of P50, but differed for P12 and P88. All three species were quite vulnerable to embolism, with P50 of -1.9 MPa (C. virgata), -2.3 MPa (C. congesta), and -2.4 MPa (C. parviflora). Estimates of P50, ΨTLP and ΨMD aligned with habitat moisture stress, with highest values found in the semi-riparian C. virgata. Consistent differences in P50, ΨMD and ΨTLP between species resulted in consistent hydraulic safety margins across species of 0.96 ± 0.1 MPa between ΨMD and P50, with onset of embolism occurring 0.43 ± 0.04 MPa after ΨTLP for all three species. Our study demonstrates that restio occupancy of dry environments involves more than the evolution of highly resistant xylem, suggesting that other aspects of water relations are key to understanding trait-environment relationships in this group.

Post-fire summer rainfall differentially affects reseeder and resprouter population recovery in fire-prone shrublands of South Africa.

Summer rainfall can have strong effects on post-fire mediterranean-type shrubland recovery patterns, with potentially long-lasting implications on communities. Our three-year field rainfall manipulation experiment tested post-fire survival and physiological responses of reseeders and resprouters to contrasting summer rainfall patterns in Fynbos and Renosterveld shrublands in South Africa. Climate projections are uncertain for this region but indicate that increased convective summer rainfall events could occur. We irrigated treatment plots during the hottest summer months (i.e. Jan, Feb, March) to contrast the naturally dry summer conditions. This allowed for assessments of the potential limiting effects of summer drought on post-fire vegetation recovery and the responsiveness of vegetation to moisture inputs during this time. Natural summer droughts led to leaf dehydration, reduced photosynthesis and reduced photosynthetic capacity. This had a particularly severe effect on reseeders during the first summer after fire leading to high mortality rates. Summer irrigations strongly reduced levels of reseeder stress and mortality. Resprouters in both vegetation types were physiologically less sensitive to rainfall patterns and showed little drought-related mortality. Comparisons of final population sizes with emergence and survival patterns showed that summer rainfall during the first summer after fire had the potential to strongly alter reseeder population sizes. The physiological sensitivity of plants to summer rainfall patterns was higher in shrubland communities occurring on fine-textured, moderately fertile soils (e.g. Renosterveld). Shrublands occurring on sandy, nutrient-poor soils (e.g. Fynbos) were remarkably insensitive to summer drought after the first summer with lower irrigation responses. Our study demonstrated the potential for variation in post-fire summer rainfall to strongly affect reseeder and resprouter population recovery patterns.

Diverse functional responses to drought in a Mediterranean-type shrubland in South Africa.

• Mediterranean-type ecosystems contain 20% of all vascular plant diversity on Earth and have been identified as being particularly threatened by future increases in drought. Of particular concern is the Cape Floral Region of South Africa, a global biodiversity hotspot, yet there are limited experimental data to validate predicted impacts on the flora. In a field rainout experiment, we tested whether rooting depth and degree of isohydry or anisohydry could aid in the functional classification of drought responses across diverse growth forms. • We imposed a 6-month summer drought, for 2 yr, in a mountain fynbos shrubland. We monitored a suite of parameters, from physiological traits to morphological outcomes, in seven species comprising the three dominant growth forms (deep-rooted proteoid shrubs, shallow-rooted ericoid shrubs and graminoid restioids). • There was considerable variation in drought response both between and within the growth forms. The shallow-rooted, anisohydric ericoid shrubs all suffered considerable reductions in growth and flowering and increased mortality. By contrast, the shallow-rooted, isohydric restioids and deep-rooted, isohydric proteoid shrubs were largely unaffected by the drought. • Rooting depth and degree of iso/anisohydry allow a first-order functional classification of drought response pathways in this flora. Consideration of additional traits would further refine this approach.

Spectral analysis software improves confidence in plant and soil water stable isotope analyses performed by isotope ratio infrared spectroscopy (IRIS).

Previous studies have demonstrated the potential for large errors to occur when analyzing waters containing organic contaminants using isotope ratio infrared spectroscopy (IRIS). In an attempt to address this problem, IRIS manufacturers now provide post-processing spectral analysis software capable of identifying samples with the types of spectral interference that compromises their stable isotope analysis. Here we report two independent tests of this post-processing spectral analysis software on two IRIS systems, OA-ICOS (Los Gatos Research Inc.) and WS-CRDS (Picarro Inc.). Following a similar methodology to a previous study, we cryogenically extracted plant leaf water and soil water and measured the δ(2)H and δ(18)O values of identical samples by isotope ratio mass spectrometry (IRMS) and IRIS. As an additional test, we analyzed plant stem waters and tap waters by IRMS and IRIS in an independent laboratory. For all tests we assumed that the IRMS value represented the "true" value against which we could compare the stable isotope results from the IRIS methods. Samples showing significant deviations from the IRMS value (>2σ) were considered to be contaminated and representative of spectral interference in the IRIS measurement. Over the two studies, 83% of plant species were considered contaminated on OA-ICOS and 58% on WS-CRDS. Post-analysis, spectra were analyzed using the manufacturer's spectral analysis software, in order to see if the software correctly identified contaminated samples. In our tests the software performed well, identifying all the samples with major errors. However, some false negatives indicate that user evaluation and testing of the software are necessary. Repeat sampling of plants showed considerable variation in the discrepancies between IRIS and IRMS. As such, we recommend that spectral analysis of IRIS data must be incorporated into standard post-processing routines. Furthermore, we suggest that the results from spectral analysis be included when reporting stable isotope data from IRIS.

Gender-specific patterns of aboveground allocation, canopy conductance and water use in a dominant riparian tree species: Acer negundo.

Acer negundo Sarg. (box elder) is a dioecious tree species that dominates riparian systems at mid elevations throughout the southwest and Intermountain West of the United States. Previous studies have shown that female A. negundo trees occur at higher frequencies along stream margins, whereas males occur at higher frequencies in drier microsites. To better understand the adaptive significance of sex ratio biases and their impact on the ecohydrology of riparian ecosystems, we examined whole-plant water relations and hydraulic properties of mature male and female A. negundo trees occurring within 1 m of a perennial stream channel. We hypothesized that (1) females would have significantly greater canopy water fluxes than males (particularly during periods of seed production: May-June), and (2) xylem in females is more hydraulically efficient but more vulnerable to cavitation than xylem in males. Mean sap flux density (J(s)) during the early growing season (May and June) was 43% higher in female trees than in male trees (n = 6 and 7 trees respectively, P < 0.0001). Mean J(s) in July and August remained 17% higher in females than in males (P = 0.0009). Mean canopy stomatal conductance per unit leaf area (g(s,leaf)) in May and June was on average 140% higher in females than in males (P < 0.0001). Mean g(s,leaf) in July and August remained 69% higher in female trees than in male trees (P < 0.0001). Canopy stomatal conductance scaled to basal area was 90 and 31% higher in females relative to males during May-June and July-August, respectively (P < 0.0001 during both periods). Conversely, there were no apparent differences in either branch hydraulic conductance or branch xylem cavitation vulnerability between genders. These results improve our capacity to describe the adaptive forces that shape the spatial distribution of male and female trees in dioecious species, and their consequences for ecohydrological processes in riparian ecosystems.

Transpiration and hydraulic strategies in a piñon-juniper woodland.

Anthropogenic climate change is likely to alter the patterns of moisture availability globally. The consequences of these changes on species distributions and ecosystem function are largely unknown, but possibly predictable based on key ecophysiological differences among currently coexisting species. In this study, we examined the environmental and biological controls on transpiration from a piñon-juniper (Pinus edulis-Juniperus osteosperma) woodland in southern Utah, USA. The potential for climate-change-associated shifts in moisture inputs could play a critical role in influencing the relative vulnerabilities of piñons and junipers to drought and affecting management decisions regarding the persistence of this dominant landscape type in the Intermountain West. We aimed to assess the sensitivity of this woodland to seasonal variations in moisture and to mechanistically explain the hydraulic strategies of P. edulis and J. osteosperma through the use of a hydraulic transport model. Transpiration from the woodland was highly sensitive to variations in seasonal moisture inputs. There were two distinct seasonal pulses of transpiration: a reliable spring pulse supplied by winter-derived precipitation, and a highly variable summer pulse supplied by monsoonal precipitation. Transpiration of P. edulis and J. osteosperma was well predicted by a mechanistic hydraulic transport model (R2 = 0.83 and 0.92, respectively). Our hydraulic model indicated that isohydric regulation of water potential in P. edulis minimized xylem cavitation during drought, which facilitated drought recovery (94% of pre-drought water uptake) but came at the cost of cessation of gas exchange for potentially extended periods. In contrast, the anisohydric J. osteosperma was able to maintain gas exchange at lower water potentials than P. edulis but experienced greater cavitation over the drought and showed a lesser degree of post-drought recovery (55% of pre-drought uptake). As a result, these species should be differentially affected by shifts in the frequency, duration, and intensity of drought. Our results highlight the sensitivity of this woodland type to potential climate-change-associated shifts in seasonal moisture patterns and demonstrate the utility of mechanistic hydraulic models in explaining differential responses of coexisting species to drought.

Differential summer water use by Pinus edulis and Juniperus osteosperma reflects contrasting hydraulic characteristics.

Previous studies of pinyon-juniper woodlands show that Pinus edulis Engelm. makes better use of soil water from summer precipitation pulses than does co-occurring Juniperus osteosperma (Torr.) Little. To investigate the basis of this difference, we examined seasonal variation in cavitation and hydraulic conductance. Pinus edulis remained isohydric over the growing season. Minimum water potentials never fell below -2.3 MPa, and the extent of xylem cavitation remained near constant during the dry season. In contrast, J. osteosperma was anisohydric, reaching water potentials as low as -6.9 MPa, and experiencing progressively greater xylem cavitation as the dry season progressed despite having more cavitation-resistant xylem than P. edulis. We conducted an irrigation experiment to observe the responses of the study species to a summer pulse of water. Although sap flow increased in both species in response to the 25-mm irrigation pulse, only J. osteosperma responded to the 10-mm pulse. This was inconsistent with the response of P. edulis to light rain events and may have been due to a difference in the distribution of irrigation water and rain water between the under- and between-canopy areas. Whole-plant conductance increased following the 25-mm irrigation in P. edulis but remained constant in J. osteosperma. We hypothesized that this difference was caused, in part, by differential refilling of embolized xylem. Area specific hydraulic conductivity was 66% higher in roots of irrigated P. edulis trees relative to roots of control trees 3 days after the 25-mm irrigation (t = 2.14, P = 0.02, df = 16). There was no change in hydraulic conductivity of the roots of J. osteosperma or in the stems of either species. Our results indicate that the response to an irrigation pulse in P. edulis depended on cavitation avoidance in stems and the reversal of cavitation in roots, resulting in increased whole-plant conductance and water uptake. In contrast, J. osteosperma failed to exploit light summer rain events but was able to extract deep soil water at low water potentials.

Population structure, physiology and ecohydrological impacts of dioecious riparian tree species of western North America.

The global water cycle is intimately linked to vegetation structure and function. Nowhere is this more apparent than in the arid west where riparian forests serve as ribbons of productivity in otherwise mostly unproductive landscapes. Dioecy is common among tree species that make up western North American riparian forests. There are intrinsic physiological differences between male and female dioecious riparian trees that may influence population structure (i.e., the ratio of male to female trees) and impact ecohydrology at large scales. In this paper, we review the current literature on sex ratio patterns and physiology of dioecious riparian tree species. Then develop a conceptual framework of the mechanisms that underlie population structure of dominant riparian tree species. Finally, we identify linkages between population structure and ecohydrological processes such as evapotranspiration and streamflow. A more thorough understanding of the mechanisms that underlie population structure of dominant riparian tree species will enable us to better predict global change impacts on vegetation and water cycling at multiple scales.

Seasonal variations in moisture use in a piñon-juniper woodland.

In water-limited environments of the intermountain region of North America, summer precipitation may play a role in the structure and function of aridland communities and ecosystems. This study examined the potential reliance on summer precipitation of two widespread, coexisting woody species in the southwestern United States, Pinus edulis Englmn. (Colorado piñon) and Juniperus osteosperma (Torr) Little (Utah juniper). The current distributions of P. edulis and J. osteosperma are highly suggestive of different dependencies on summer rainfall. We hypothesized that P. edulis was dependent on summer precipitation, utilizing summer precipitation even during extremely dry summers, whereas J. osteosperma was not dependent, using summer precipitation only when amounts were above some minimum threshold. Using sap flux and stable isotopic methods to assess seasonal water sources and water use efficiency, we examined the response of these two species to seasonal variations in moisture at a site located near the northern limits of the North American monsoon. Both sap flux and isotopic results indicated that P. edulis was responsive to summer rain, while J. osteosperma was not. Following summer rain events, sap flux density increased in P. edulis for several days, but not in J. osteosperma. Isotopic evidence indicated that P. edulis took up summer-derived moisture to a greater extent than J. osteosperma. Values of the natural abundance stable isotope ratio of carbon of leaf soluble carbohydrates increased over the summer for P. edulis, indicative of assimilation at higher water use efficiency, but were invariant for J. osteosperma. Our results supported the hypothesis that P. edulis and J. osteosperma are differentially sensitive to summer precipitation and are discussed in the light of potential changes in the seasonality of precipitation associated with climate change.

Turnover of carbon isotopes in tail hair and breath CO2 of horses fed an isotopically varied diet.

Temporal stable isotope records derived from animal tissues are increasingly studied to determine dietary and climatic histories. Despite this, the turnover times governing rates of isotope equilibration in specific tissues following a dietary isotope change are poorly known. The dietary isotope changes recorded in the hair and blood bicarbonate of two adult horses in this study are found to be successfully described by a model having three exponential isotope pools. For horse tail hair, the carbon isotope response observed following a dietary change from a C3 to a C4 grass was consistent with a pool having a very fast turnover rate ( t1/2 approximately 0.5 days) that made up approximately 41% of the isotope signal, a pool with an intermediate turnover rate ( t1/2 approximately 4 days) that comprised approximately 15% of the isotope signal, and a pool with very slow turnover rate ( t1/2 approximately 140 days) that made up approximately 44% of the total isotope signal. The carbon isotope signature of horse blood bicarbonate, in contrast, had a different isotopic composition, with approximately 67% of the isotope signal coming from a fast turnover pool ( t1/2 0.2 days), approximately 17% from a pool with an intermediate turnover rate ( t1/2 approximately 3 days) and approximately 16% from a pool with a slow turnover rate ( t1/2 approximately 50 days). The constituent isotope pools probably correspond to one exogenous and two endogenous sources. The exogenous source equates to our fast turnover pool, and the pools with intermediate and slow turnover rates are thought to derive from the turnover of metabolically active tissues and relatively inactive tissues within the body, respectively. It seems that a greater proportion of the amino acids available for hair synthesis come from endogenous sources compared to the compounds undergoing cellular catabolism in the body. Consequently, the isotope composition of blood bicarbonate appears to be much more responsive to dietary isotope changes, whereas the amino acids in the blood exhibit considerable isotopic inertia.

The forkhead protein Fkh2 is a component of the yeast cell cycle transcription factor SFF.

In the yeast Saccharomyces cerevisiae, the MADS-box protein Mcm1, which is highly related to mammalian SRF (serum response factor), forms a ternary complex with SFF (Swi five factor) to regulate the cell cycle expression of genes such as SWI5, CLB2 and ACE2. Here we show that the forkhead protein Fkh2 is a component of SFF and is essential for ternary complex formation on the SWI5 and ACE2 promoters. Fkh2 is essential for the correct cell cycle periodicity of SWI5 and CLB2 gene expression and is phosphorylated with a timing that is consistent with a role in this expression. Furthermore, investigation of the relationship between Fkh2 and a related forkhead protein Fkh1 demonstrates that these proteins act in overlapping pathways to regulate cell morphology and cell separation. This is the first example of a eukaryotic transcription factor complex containing both a MADS-box and a forkhead protein, and it has important implications for the regulation of mammalian gene expression.

Structural and functional conservation at the boundaries of the chicken beta-globin domain.

We show that the 3' boundary of the chicken beta-globin locus bears striking structural similarities to the 5' boundary. In erythroid cells a clear transition in DNase I sensitivity of chromatin at the 3' end of the locus is observed, the location of this transition is marked by a constitutive DNase I hypersensitive site (HS), and DNA spanning this site has the enhancer-blocking capacity of an insulator. This HS contains a binding site for the transcription factor CTCF. As in the case of the 5' insulator, the CTCF site is both necessary and sufficient for the enhancer-blocking activity of the 3' boundary. The position of this insulator is consistent with our proposal that it may function to maintain the distinct regulatory programs of the globin genes and their closely appended 3' neighbor, an odorant receptor gene. We conclude that both boundaries of the chicken beta-globin domain are capable of playing functionally similar roles and that the same protein is a necessary component of the molecular mechanism through which these boundaries are defined.

The protein CTCF is required for the enhancer blocking activity of vertebrate insulators.

An insulator is a DNA sequence that can act as a barrier to the influences of neighboring cis-acting elements, preventing gene activation, for example, when located between an enhancer and a promoter. We have identified a 42 bp fragment of the chicken beta-globin insulator that is both necessary and sufficient for enhancer blocking activity in human cells. We show that this sequence is the binding site for CTCF, a previously identified eleven-zinc finger DNA-binding protein that is highly conserved in vertebrates. CTCF sites are present in all of the vertebrate enhancer-blocking elements we have examined. We suggest that directional enhancer blocking by CTCF is a conserved component of gene regulation in vertebrates.

MADS-box transcription factors adopt alternative mechanisms for bending DNA.

Transcription factor-induced DNA bending is important in determining local promoter architecture and it is thought to be a key determinant of their function. The human MADS-box transcription factors serum response factor and MEF2A exhibit different propensities to bend their binding sites. Here, we have investigated the ability of several family members from different species to bend DNA and the molecular mechanisms underlying this process. Differential DNA bending is observed in yeast and plant MADS-box proteins. Like MEF2A, the yeast proteins Rlm1 and Smp1 exhibit low DNA bending propensities. A comparison of serum response factor and SQUA reveals that the basic mechanisms of DNA bending appear to be conserved between these proteins, although several key differences do exist. In contrast to serum response factor, SQUA bends DNA in a DNA sequence-dependent manner. In both proteins, protein-DNA contacts made between residues in the beta-loop and the N-terminal end of the recognition helices in the MADS-box are the major determinants of DNA bending. However, although residues which are involved in DNA bending are predicted to be located in similar positions in their tertiary structures, different residues dictate bending by each protein. Further complexities are uncovered in the links between the DNA bending propensity and the binding specificity. In combination with structural studies, our results provide a model to explain how differential bending by MADS-box proteins is achieved at the molecular level and provide insights into how this might affect their biological function.

DNA binding and dimerisation determinants of Antirrhinum majus MADS-box transcription factors.

Members of the MADS-box family of transcription factors are found in eukaryotes ranging from yeast to humans. In plants, MADS-box proteins regulate several developmental processes including flower, fruit and root development. We have investigated the DNA-binding mechanisms used by four such proteins in Antirrhinum majus, SQUA, PLE, DEF and GLO. SQUA differs from the characterised mammalian and yeast MADS-box proteins as it can efficiently bind two different classes of DNA-binding site. SQUA induces bending of these binding sites and the sequence of the site plays a role in determining the magnitude of these bends. Similarly, PLE and DEF/GLO induce DNA bending although the direction of the resulting bends differ. Finally, we demonstrate that the MADS-box and I-domains are sufficient for homodimer formation by SQUA. However, the K-box in SQUA can also act as an oligomerisation motif and in the full-length protein, the K-box plays a different role in mediating dimerisation in the context of SQUA homodimers or heterodimers with PLE. Together these results contribute significantly to our understanding of the function of SQUA and other plant MADS-box proteins at the molecular level.

Interaction of transcription factors with serum response factor. Identification of the Elk-1 binding surface.

Serum response elements (SREs) play important roles in transforming extracellular signals into specific nuclear responses. The SRE-binding protein, serum response factor (SRF), plays a pivotal role in this process. Several transcription factors have been shown to interact with SRF and thereby create distinct complexes with different regulatory potentials. The ETS domain transcription factor Elk-1 is one such protein and serves to integrate distinct mitogen-activated protein kinase cascades at SREs. Elk-1 uses a short hydrophobic surface presented on the surface of an alpha-helix to interact with SRF. In this study we have used site-directed mutagenesis to identify residues in SRF that comprise the Elk-1 binding surface. The Elk-1 binding surface is composed of residues that lie on a hydrophobic surface-exposed groove located at the junction of the MADS box and C-terminal SAM motif. Different residues are implicated in interactions between SRF and the transcription factor Fli-1, indicating that although some overlap with the Elk-1 binding surface occurs, their interaction surfaces on SRF are distinct. Our data are consistent with the hypothesis that the SRF DNA-binding domain acts as docking site for multiple transcription factors that can bind to small surface-exposed patches within this domain.

DNA binding by MADS-box transcription factors: a molecular mechanism for differential DNA bending.

The serum response factor (SRF) and myocyte enhancer factor 2A (MEF2A) represent two human members of the MADS-box transcription factor family. Each protein has a distinct biological function which is reflected by the distinct specificities of the proteins for coregulatory protein partners and DNA-binding sites. In this study, we have investigated the mechanism of DNA binding utilized by these two related transcription factors. Although SRF and MEF2A belong to the same family and contain related DNA-binding domains, their DNA-binding mechanisms differ in several key aspects. In contrast to the dramatic DNA bending induced by SRF, MEF2A induces minimal DNA distortion. A combination of loss- and gain-of-function mutagenesis identified a single amino acid residue located at the N terminus of the recognition helices as the critical mediator of this differential DNA bending. This residue is also involved in determining DNA-binding specificity, thus indicating a link between DNA bending and DNA-binding specificity determination. Furthermore, different basic residues within the putative recognition alpha-helices are critical for DNA binding, and the role of the C-terminal extensions to the MADS box in dimerization between SRF and MEF2A also differs. These important differences in the molecular interactions of SRF and MEF2A are likely to contribute to their differing roles in the regulation of specific gene transcription.

Incorporating psychological debriefing techniques within a brief group psychotherapy programme for the treatment of post-traumatic stress disorder.

BACKGROUND: The Royal Air Force Wroughton Post-Traumatic Stress Disorder (PTSD) Rehabilitation Programme is described. It comprised a 12-day structured in-patient 'course' of group psychotherapy and day-case group follow-up sessions over a one-year period. Psychological debriefing was the main therapeutic technique employed. METHOD: This is a 'before and after' open outcome study. A comprehensive assessment protocol confirmed the presence and severity of PTSD and measured co-morbid psychopathological status, occupational and social function longitudinally. RESULTS: A highly significant global response to treatment is demonstrated in the 34 subjects included in the study, with 85.3% not fulfilling the DSM-III-R criteria for PTSD at one year after treatment. CONCLUSIONS: Further controlled studies assessing the value of psychological debriefing techniques in the treatment of established PTSD are required.