Joint effects of climate, tree size, and year on annual tree growth derived from tree-ring records of ten globally distributed forests.

Tree rings provide an invaluable long-term record for understanding how climate and other drivers shape tree growth and forest productivity. However, conventional tree-ring analysis methods were not designed to simultaneously test effects of climate, tree size, and other drivers on individual growth. This has limited the potential to test ecologically relevant hypotheses on tree growth sensitivity to environmental drivers and their interactions with tree size. Here, we develop and apply a new method to simultaneously model nonlinear effects of primary climate drivers, reconstructed tree diameter at breast height (DBH), and calendar year in generalized least squares models that account for the temporal autocorrelation inherent to each individual tree's growth. We analyze data from 3811 trees representing 40 species at 10 globally distributed sites, showing that precipitation, temperature, DBH, and calendar year have additively, and often interactively, influenced annual growth over the past 120 years. Growth responses were predominantly positive to precipitation (usually over ≥3-month seasonal windows) and negative to temperature (usually maximum temperature, over ≤3-month seasonal windows), with concave-down responses in 63% of relationships. Climate sensitivity commonly varied with DBH (45% of cases tested), with larger trees usually more sensitive. Trends in ring width at small DBH were linked to the light environment under which trees established, but basal area or biomass increments consistently reached maxima at intermediate DBH. Accounting for climate and DBH, growth rate declined over time for 92% of species in secondary or disturbed stands, whereas growth trends were mixed in older forests. These trends were largely attributable to stand dynamics as cohorts and stands age, which remain challenging to disentangle from global change drivers. By providing a parsimonious approach for characterizing multiple interacting drivers of tree growth, our method reveals a more complete picture of the factors influencing growth than has previously been possible.

The effect of species, size, and fire intensity on tree mortality within a catastrophic bushfire complex.

Infrequent, high-intensity disturbances can have profound impacts on forested landscapes, changing forest structure and altering relative species abundance. However, due to their rarity and the logistical challenges of directly observing such extreme events, both the spatial variability of disturbance intensity and the species-specific responses to this variability are poorly understood. We used observed patterns of mortality across a fire severity gradient following the 2009 Black Saturday fires in southeastern Australia to simultaneously estimate (1) species- and size-specific susceptibility to fire-induced mortality and (2) fire intensity. We found broad variation in patterns of fire susceptibility among the 10 tree species (five eucalypts and five non-eucalypts) sufficiently abundant for analysis. Among the eucalypts, Eucalyptus obliqua was the most resistant to fire-induced mortality, with trees of ~25 cm DBH having a 50% probability of surviving even the most intense fires. In contrast, E. regnans had 100% mortality across all size classes when subjected to high-intensity fire. Basal resprouting occurred in six of the study species and, when accounted for, fundamentally changed the mortality profile of these species, highlighting the importance of resprouting as an adaptation to fire in these landscapes. In particular, the two iconic cool temperate rainforest species (Nothofagus cunninghami and Atherosperma moschatum) were strong resprouters (~45% of individuals were able to resprout after being top-killed by fire). We also found evidence for compositional shifts in regeneration above threshold values of fire intensity in cool temperate rainforest and mixed forest sites, both of which have important conservation values within these landscapes. The observed patterns of species- and size-specific susceptibility to fire-induced mortality may be used to anticipate changes in forest structure and composition in the future. In addition, they may also help guide forest management strategies that reduce the length of time individual trees are exposed to potentially lethal fires, thereby increasing the resilience of these forests to future fires.

Elucidating the structural basis for differing enzyme inhibitor potency by cryo-EM.

Histidine biosynthesis is an essential process in plants and microorganisms, making it an attractive target for the development of herbicides and antibacterial agents. Imidazoleglycerol-phosphate dehydratase (IGPD), a key enzyme within this pathway, has been biochemically characterized in both Saccharomyces cerevisiae (Sc_IGPD) and Arabidopsis thaliana (At_IGPD). The plant enzyme, having been the focus of in-depth structural analysis as part of an inhibitor development program, has revealed details about the reaction mechanism of IGPD, whereas the yeast enzyme has proven intractable to crystallography studies. The structure-activity relationship of potent triazole-phosphonate inhibitors of IGPD has been determined in both homologs, revealing that the lead inhibitor (C348) is an order of magnitude more potent against Sc_IGPD than At_IGPD; however, the molecular basis of this difference has not been established. Here we have used single-particle electron microscopy (EM) to study structural differences between the At and Sc_IGPD homologs, which could influence the difference in inhibitor potency. The resulting EM maps at ∼3 Šare sufficient to de novo build the protein structure and identify the inhibitor binding site, which has been validated against the crystal structure of the At_IGPD/C348 complex. The structure of Sc_IGPD reveals that a 24-amino acid insertion forms an extended loop region on the enzyme surface that lies adjacent to the active site, forming interactions with the substrate/inhibitor binding loop that may influence inhibitor potency. Overall, this study provides insights into the IGPD family and demonstrates the power of using an EM approach to study inhibitor binding.

The molecular basis of endolytic activity of a multidomain alginate lyase from Defluviitalea phaphyphila, a representative of a new lyase family, PL39.

Alginate is a polymer containing two uronic acid epimers, ß-d-mannuronate (M) and α-l-guluronate (G), and is a major component of brown seaweed that is depolymerized by alginate lyases. These enzymes have diverse specificity, cleaving the chain with endo- or exotype activity and with differential selectivity for the sequence of M or G at the cleavage site. Dp0100 is a 201-kDa multimodular, broad-specificity endotype alginate lyase from the marine thermophile Defluviitalea phaphyphila, which uses brown algae as a carbon source, converting it to ethanol, and bioinformatics analysis suggested that its catalytic domain represents a new polysaccharide lyase family, PL39. The structure of the Dp0100 catalytic domain, determined at 2.07 Å resolution, revealed that it comprises three regions strongly resembling those of the exotype lyase families PL15 and PL17. The conservation of key catalytic histidine and tyrosine residues belonging to the latter suggests these enzymes share mechanistic similarities. A complex of Dp0100 with a pentasaccharide, M5, showed that the oligosaccharide is located in subsites -2, -1, +1, +2, and +3 in a long, deep canyon open at both ends, explaining the endotype activity of this lyase. This contrasted with the hindered binding sites of the exotype enzymes, which are blocked such that only one sugar moiety can be accommodated at the -1 position in the catalytic site. The biochemical and structural analyses of Dp0100, the first for this new class of endotype alginate lyases, have furthered our understanding of the structure-function and evolutionary relationships within this important class of enzymes.

Conserved residues in Ycf54 are required for protochlorophyllide formation in Synechocystis sp. PCC 6803.

Chlorophylls (Chls) are modified tetrapyrrole molecules, essential for photosynthesis. These pigments possess an isocyclic E ring formed by the Mg-protoporphyrin IX monomethylester cyclase (MgPME-cyclase). We assessed the in vivo effects of altering seven highly conserved residues within Ycf54, which is required for MgPME-cyclase activity in the cyanobacterium SynechocystisSynechocystis strains harbouring the Ycf54 alterations D39A, F40A and R82A were blocked to varying degrees at the MgPME-cyclase step, whereas the A9G mutation reduced Ycf54 levels by ∼75%. Wild-type (WT) levels of the cyclase subunit CycI are present in strains with D39A and F40A, but these strains have lowered cellular Chl and photosystem accumulation. CycI is reduced by ∼50% in A9G and R82A, but A9G has no perturbations in Chl or photosystem accumulation, whilst R82A contains very little Chl and few photosystems. When FLAG tagged and used as bait in pulldown experiments, the three mutants D39A, F40A and R82A were unable to interact with the MgPME-cyclase component CycI, whereas A9G pulled down a similar level of CycI as WT Ycf54. These observations suggest that a stable interaction between CycI and Ycf54 is required for unimpeded Pchlide biosynthesis. Crystal structures of the WT, A9G and R82A Ycf54 proteins were solved and analysed to investigate the structural effects of these mutations. A loss of the local hydrogen bonding network and a reversal in the surface charge surrounding residue R82 are probably responsible for the functional differences observed in the R82A mutation. We conclude that the Ycf54 protein must form a stable interaction with CycI to promote optimal Pchlide biosynthesis.

Mirror-Image Packing Provides a Molecular Basis for the Nanomolar Equipotency of Enantiomers of an Experimental Herbicide.

Programs of drug discovery generally exploit one enantiomer of a chiral compound for lead development following the principle that enantiomer recognition is central to biological specificity. However, chiral promiscuity has been identified for a number of enzyme families, which have shown that mirror-image packing can enable opposite enantiomers to be accommodated in an enzyme's active site. Reported here is a series of crystallographic studies of complexes between an enzyme and a potent experimental herbicide whose chiral center forms an essential part of the inhibitor pharmacophore. Initial studies with a racemate at 1.85 Šresolution failed to identify the chirality of the bound inhibitor, however, by extending the resolution to 1.1 Šand by analyzing high-resolution complexes with the enantiopure compounds, we determined that both enantiomers make equivalent pseudosymmetric interactions in the active site, thus mimicking an achiral reaction intermediate.

Reforestation with native mixed-species plantings in a temperate continental climate effectively sequesters and stabilizes carbon within decades.

Reforestation has large potential for mitigating climate change through carbon sequestration. Native mixed-species plantings have a higher potential to reverse biodiversity loss than do plantations of production species, but there are few data on their capacity to store carbon. A chronosequence (5-45 years) of 36 native mixed-species plantings, paired with adjacent pastures, was measured to investigate changes to stocks among C pools following reforestation of agricultural land in the medium rainfall zone (400-800 mm yr(-1)) of temperate Australia. These mixed-species plantings accumulated 3.09 ± 0.85 t C ha(-1) yr(-1) in aboveground biomass and 0.18 ± 0.05 t C ha(-1) yr(-1) in plant litter, reaching amounts comparable to those measured in remnant woodlands by 20 years and 36 years after reforestation respectively. Soil C was slower to increase, with increases seen only after 45 years, at which time stocks had not reached the amounts found in remnant woodlands. The amount of trees (tree density and basal area) was positively associated with the accumulation of carbon in aboveground biomass and litter. In contrast, changes to soil C were most strongly related to the productivity of the location (a forest productivity index and soil N content in the adjacent pasture). At 30 years, native mixed-species plantings had increased the stability of soil C stocks, with higher amounts of recalcitrant C and higher C:N ratios than their adjacent pastures. Reforestation with native mixed-species plantings did not significantly change the availability of macronutrients (N, K, Ca, Mg, P, and S) or micronutrients (Fe, B, Mn, Zn, and Cu), content of plant toxins (Al, Si), acidity, or salinity (Na, electrical conductivity) in the soil. In this medium rainfall area, native mixed-species plantings provided comparable rates of C sequestration to local production species, with the probable additional benefit of providing better quality habitat for native biota. These results demonstrate that reforestation using native mixed-species plantings is an effective alternative for carbon sequestration to standard monocultures of production species in medium rainfall areas of temperate continental climates, where they can effectively store C, convert C into stable pools and provide greater benefits for biodiversity.

Temporal variability of forest communities: empirical estimates of population change in 4000 tree species.

Long-term surveys of entire communities of species are needed to measure fluctuations in natural populations and elucidate the mechanisms driving population dynamics and community assembly. We analysed changes in abundance of over 4000 tree species in 12 forests across the world over periods of 6-28 years. Abundance fluctuations in all forests are large and consistent with population dynamics models in which temporal environmental variance plays a central role. At some sites we identify clear environmental drivers, such as fire and drought, that could underlie these patterns, but at other sites there is a need for further research to identify drivers. In addition, cross-site comparisons showed that abundance fluctuations were smaller at species-rich sites, consistent with the idea that stable environmental conditions promote higher diversity. Much community ecology theory emphasises demographic variance and niche stabilisation; we encourage the development of theory in which temporal environmental variance plays a central role.

Temperature and rainfall strongly drive temporal growth variation in Asian tropical forest trees.

Climate change effects on growth rates of tropical trees may lead to alterations in carbon cycling of carbon-rich tropical forests. However, climate sensitivity of broad-leaved lowland tropical trees is poorly understood. Dendrochronology (tree-ring analysis) provides a powerful tool to study the relationship between tropical tree growth and annual climate variability. We aimed to establish climate-growth relationships for five annual-ring forming tree species, using ring-width data from 459 canopy and understory trees from a seasonal tropical forest in western Thailand. Based on 183/459 trees, chronologies with total lengths between 29 and 62 years were produced for four out of five species. Bootstrapped correlation analysis revealed that climate-growth responses were similar among these four species. Growth was significantly negatively correlated with current-year maximum and minimum temperatures, and positively correlated with dry-season precipitation levels. Negative correlations between growth and temperature may be attributed to a positive relationship between temperature and autotrophic respiration rates. The positive relationship between growth and dry-season precipitation levels likely reflects the strong water demand during leaf flush. Mixed-effect models yielded results that were consistent across species: a negative effect of current wet-season maximum temperatures on growth, but also additive positive effects of, for example, prior dry-season maximum temperatures. Our analyses showed that annual growth variability in tropical trees is determined by a combination of both temperature and precipitation variability. With rising temperature, the predominantly negative relationship between temperature and growth may imply decreasing growth rates of tropical trees as a result of global warming.

Crystallization and preliminary crystallographic analysis of a surface antigen glycoprotein, SAG19, from Eimeria tenella.

Coccidiosis in chickens is caused by the apicomplexan parasite Eimeria tenella and is thought to involve a role for a superfamily of more than 20 cysteine-rich surface antigen glycoproteins (SAGs) in host-parasite interactions. A representative member of the family, SAG19, has been overexpressed in Escherichia coli, purified and crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. Crystals of SAG19 diffracted to beyond 1.50 Å resolution and belonged to space group I4, with unit-cell parameters a = b = 108.2, c = 37.5 Å. Calculation of possible values of VM suggests that there is a single molecule in the asymmetric unit.

Cloning, purification, crystallization and preliminary X-ray analysis of the Burkholderia pseudomallei L1 ribosomal protein.

The gene encoding the L1 ribosomal protein from Burkholderia pseudomallei strain D286 has been cloned into the pETBLUE-1 vector system, overexpressed in Escherichia coli and purified. Crystals of the native protein were grown by the hanging-drop vapour-diffusion technique using PEG 3350 as a precipitant and diffracted to beyond 1.65 Å resolution. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 53.6, b = 127.1, c = 31.8 Å and with a single molecule in the asymmetric unit.

Crystallization and preliminary X-ray analysis of the receiver domain of a putative response regulator, BPSL0128, from Burkholderia pseudomallei.

bpsl0128, a gene encoding a putative response regulator from Burkholderia pseudomallei strain D286, has been cloned into a pETBLUE-1 vector system, overexpressed in Escherichia coli and purified. The full-length protein is degraded during purification to leave a fragment corresponding to the putative receiver domain, and crystals of this protein that diffracted to beyond 1.75 Šresolution have been grown by the hanging-drop vapour-diffusion technique using PEG 6000 as the precipitant. The crystals belonged to one of the enantiomorphic pair of space groups P3(1)21 and P3(2)21, with unit-cell parameters a = b = 65.69, c = 105.01 Šand either one or two molecules in the asymmetric unit.

Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily.

Despite being the subject of intensive investigations, many aspects of the mechanism of the zinc-dependent medium chain alcohol dehydrogenase (MDR) superfamily remain contentious. We have determined the high-resolution structures of a series of binary and ternary complexes of glucose dehydrogenase, an MDR enzyme from Haloferax mediterranei. In stark contrast to the textbook MDR mechanism in which the zinc ion is proposed to remain stationary and attached to a common set of protein ligands, analysis of these structures reveals that in each complex, there are dramatic differences in the nature of the zinc ligation. These changes arise as a direct consequence of linked movements of the zinc ion, a zinc-bound bound water molecule, and the substrate during progression through the reaction. These results provide evidence for the molecular basis of proton traffic during catalysis, a structural explanation for pentacoordinate zinc ion intermediates, a unifying view for the observed patterns of metal ligation in the MDR family, and highlight the importance of dynamic fluctuations at the metal center in changing the electrostatic potential in the active site, thereby influencing the proton traffic and hydride transfer events.

Molecular basis of specificity and deamidation of eIF4A by Burkholderia Lethal Factor 1.

Burkholderia pseudomallei lethal factor 1 (BLF1) exhibits site-specific glutamine deamidase activity against the eukaryotic RNA helicase, eIF4A, thereby blocking mammalian protein synthesis. The structure of a complex between BLF1 C94S and human eIF4A shows that the toxin binds in the cleft between the two RecA-like eIF4A domains forming interactions with residues from both and with the scissile amide of the target glutamine, Gln339, adjacent to the toxin active site. The RecA-like domains adopt a radically twisted orientation compared to other eIF4A structures and the nature and position of conserved residues suggests this may represent a conformation associated with RNA binding. Comparison of the catalytic site of BLF1 with other deamidases and cysteine proteases reveals that they fall into two classes, related by pseudosymmetry, that present either the re or si faces of the target amide/peptide to the nucleophilic sulfur, highlighting constraints in the convergent evolution of their Cys-His active sites.

Cloning, purification and crystallographic analysis of a hypothetical protein, BPSL1549, from Burkholderia pseudomallei.

Burkholderia pseudomallei BPSL1549, a putative protein of unknown function, has been overexpressed in Escherichia coli, purified and subsequently crystallized by the hanging-drop vapour-diffusion method using PEG as a precipitant to give crystals with overall dimensions of 0.15 × 0.15 × 0.1 mm. Native data were collected to 1.47 Å resolution at the European Synchrotron Radiation Facility (ESRF). The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 37.1, b = 45.4, c = 111.9 Å and with a single polypeptide chain in the asymmetric unit.

Mapping canopy nitrogen-scapes to assess foraging habitat for a vulnerable arboreal folivore in mixed-species Eucalyptus forests.

Herbivore foraging decisions are closely related to plant nutritional quality. For arboreal folivores with specialized diets, such as the vulnerable greater glider (Petauroides volans), the abundance of suitable forage trees can influence habitat suitability and species occurrence. The ability to model and map foliar nitrogen would therefore enhance our understanding of folivore habitat use at finer scales. We tested whether high-resolution multispectral imagery, collected by a lightweight and low-cost commercial unoccupied aerial vehicle (UAV), could be used to predict total and digestible foliar nitrogen (N and digN) at the tree canopy level and forest stand-scale from leaf-scale chemistry measurements across a gradient of mixed-species Eucalyptus forests in southeastern Australia. We surveyed temperate Eucalyptus forests across an elevational and topographic gradient from sea level to high elevation (50-1200 m a.s.l.) for forest structure, leaf chemistry, and greater glider occurrence. Using measures of multispectral leaf reflectance and spectral indices, we estimated N and digN and mapped N and favorable feeding habitat using machine learning algorithms. Our surveys covered 17 Eucalyptus species ranging in foliar N from 0.63% to 1.92% dry matter (DM) and digN from 0.45% to 1.73% DM. Both multispectral leaf reflectance and spectral indices were strong predictors for N and digN in model cross-validation. At the tree level, 79% of variability between observed and predicted measures of nitrogen was explained. A spatial supervised classification model correctly identified 80% of canopy pixels associated with high N concentrations (≥1% DM). We developed a successful method for estimating foliar nitrogen of a range of temperate Eucalyptus species using UAV multispectral imagery at the tree canopy level and stand scale. The ability to spatially quantify feeding habitat using UAV imagery allows remote assessments of greater glider habitat at a scale relevant to support ground surveys, management, and conservation for the vulnerable greater glider across southeastern Australia.

Compound climate extremes driving recent sub-continental tree mortality in northern Australia have no precedent in recent centuries.

Compound climate extremes (CCEs) can have significant and persistent environmental impacts on ecosystems. However, knowledge of the occurrence of CCEs beyond the past ~ 50 years, and hence their ecological impacts, is limited. Here, we place the widespread 2015-16 mangrove dieback and the more recent 2020 inland native forest dieback events in northern Australia into a longer historical context using locally relevant palaeoclimate records. Over recent centuries, multiple occurrences of analogous antecedent and coincident climate conditions associated with the mangrove dieback event were identified in this compilation. However, rising sea level-a key antecedent condition-over the three decades prior to the mangrove dieback is unprecedented in the past 220 years. Similarly, dieback in inland forests and savannas was associated with a multi-decadal wetting trend followed by the longest and most intense drought conditions of the past 250 years, coupled with rising temperatures. While many ecological communities may have experienced CCEs in past centuries, the addition of new environmental stressors associated with varying aspects of global change may exceed their thresholds of resilience. Palaeoclimate compilations provide the much-needed longer term context to better assess frequency and changes in some types of CCEs and their environmental impacts.

Characterisation of a tripartite α-pore forming toxin from Serratia marcescens.

Tripartite members of the ClyA family of α-PFTs have recently been identified in a number of pathogenic Gram-negative bacteria, including the human pathogen Serratia marcescens. Structures of a Gram-negative A component and a tripartite α-PFT complete pore are unknown and a mechanism for pore formation is still uncertain. Here we characterise the tripartite SmhABC toxin from S. marcescens and propose a mechanism of pore assembly. We present the structure of soluble SmhA, as well as the soluble and pore forms of SmhB. We show that the ß-tongue soluble structure is well conserved in the family and propose two conserved latches between the head and tail domains that are broken on the soluble to pore conformational change. Using the structures of individual components, sequence analysis and docking predictions we illustrate how the A, B and C protomers would assemble on the membrane to produce a complete tripartite α-PFT pore.

The structure of a major surface antigen SAG19 from Eimeria tenella unifies the Eimeria SAG family.

In infections by apicomplexan parasites including Plasmodium, Toxoplasma gondii, and Eimeria, host interactions are mediated by proteins including families of membrane-anchored cysteine-rich surface antigens (SAGs) and SAG-related sequences (SRS). Eimeria tenella causes caecal coccidiosis in chickens and has a SAG family with over 80 members making up 1% of the proteome. We have solved the structure of a representative E. tenella SAG, EtSAG19, revealing that, despite a low level of sequence similarity, the entire Eimeria SAG family is unified by its three-layer αßα fold which is related to that of the CAP superfamily. Furthermore, sequence comparisons show that the Eimeria SAG fold is conserved in surface antigens of the human coccidial parasite Cyclospora cayetanensis but this fold is unrelated to that of the SAGs/SRS proteins expressed in other apicomplexans including Plasmodium species and the cyst-forming coccidia Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. However, despite having very different structures, Consurf analysis showed that Eimeria SAG and Toxoplasma SRS families each exhibit marked hotspots of sequence hypervariability that map to their surfaces distal to the membrane anchor. This suggests that the primary and convergent purpose of the different structures is to provide a platform onto which sequence variability can be imposed.

Structural origins of pH-dependent chemical shifts in the B1 domain of protein G.

We report chemical shifts for H(N), N, and C' nuclei in the His-tagged B1 domain of protein G (GB1) over a range of pH values from pH 2.0 to 9.0, which fit well to standard pH-dependent equations. We also report a 1.2 Å resolution crystal structure of GB1 at pH 3.0. Comparison of this crystal structure with published crystal structures at higher pHs provides details of the structural changes in GB1 associated with protonation of the carboxylate groups, in particular a conformational change in the C-terminus of the protein at low pH. An additional change described recently is not seen in the crystal structure because of crystal contacts. We show that the pH-dependent changes in chemical shifts can be almost entirely understood based on structural changes, thereby providing insight into the relationship between structure and chemical shift. In particular, we describe through-bond effects extending up to five bonds, affecting N and C' but not H(N); through-space effects of carboxylates, which fit well to a simple electric field model; and effects due to conformational change, which have a similar magnitude to many of the direct effects. Finally, we discuss cooperative effects, demonstrating a lack of cooperative unfolding in the helix, and the existence of a ß-sheet "iceberg" extending over three of the four strands. This study therefore extends the application of chemical shifts to understanding protein structure.