Protease shaving of Mycobacterium tuberculosis facilitates vaccine antigen discovery and delivery of novel cargoes to the Mtb surface.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of infectious disease death and lacks a vaccine capable of protecting adults from pulmonary TB. Studies have shown that Mtb uses a variety of mechanisms to evade host immunity. Secreted Mtb proteins such as Type VII secretion system substrates have been characterized for their ability to modulate anti-Mtb immunity; however, studies of other pathogens such as Salmonella Typhi and Staphylococcus aureus have revealed that outer membrane proteins can also interact with the innate and adaptive immune system. The Mtb outer membrane proteome has received relatively less attention due to limited techniques available to interrogate this compartment. We filled this gap by deploying protease shaving and quantitative mass spectrometry to identify Mtb outer membrane proteins which serve as nodes in the Mtb-host interaction network. These analyses revealed several novel Mtb proteins on the Mtb surface largely derived from the PE/PPE class of Mtb proteins, including PPE18, a component of a leading Mtb vaccine candidate. We next exploited the localization of PPE18 to decorate the Mtb surface with heterologous proteins and deliver these surface-engineered Mtb to the phagosome. Together, these studies reveal potential novel targets for new Mtb vaccines as well as facilitate new approaches to study difficult to study cellular compartments during infection.

Reduced tillering and dwarfing genes alter root traits and rhizo-economics in wheat.

The tiller inhibition (tin) and Reduced height (Rht) genes strongly influence the carbon partitioning and architecture of wheat shoots, but their effects on the energy economy of roots have not been examined in detail. We examined multiple root traits in three sets of near-isogenic wheat lines (NILs) that differ in the tin gene or various dwarfing gene alleles (Rht-B1b, Rht-D1b, Rht-B1c and Rht-B1b + Rht-D1b) to determine their effects on root structure, anatomy and carbon allocation. The tin gene resulted in fewer tillers but more costly roots in an extreme tin phenotype with a Banks genetic background due to increases in root-to-shoot ratio, total root length, and whole root respiration. However, this effect depended on the genetic background as tin caused both smaller shoots and roots in a different genetic background. The semi-dwarf gene Rht-B1b caused few changes to the root structure, whereas Rht-D1b, Rht-B1c and the double dwarf (Rht-B1b + Rht-D1b) decreased the root biomass. Rht-B1c reduced the energy cost of roots by increasing specific root length, increasing the volume of cortical aerenchyma and by reducing root length, number, and biomass without affecting the root-to-shoot ratio. This work informs researchers using tin and Rht genes how to modify root system architecture to suit specific environments.

Variation in leaf dark respiration among C3 and C4 grasses is associated with use of different substrates.

Measurements of respiratory properties have often been made at a single time point either during daytime using dark-adapted leaves or during nighttime. The influence of the day-night cycle on respiratory metabolism has received less attention but is crucial to understand photosynthesis and photorespiration. Here, we examined how CO2- and O2-based rates of leaf dark respiration (Rdark) differed between midday (after 30-min dark adaptation) and midnight in 8 C3 and C4 grasses. We used these data to calculate the respiratory quotient (RQ; ratio of CO2 release to O2 uptake), and assessed relationships between Rdark and leaf metabolome. Rdark was higher at midday than midnight, especially in C4 species. The day-night difference in Rdark was more evident when expressed on a CO2 than O2 basis, with the RQ being higher at midday than midnight in all species, except in rice (Oryza sativa). Metabolomic analyses showed little correlation of Rdark or RQ with leaf carbohydrates (sucrose, glucose, fructose, or starch) but strong multivariate relationships with other metabolites. The results suggest that rates of Rdark and differences in RQ were determined by several concurrent CO2-producing and O2-consuming metabolic pathways, not only the tricarboxylic acid cycle (organic acids utilization) but also the pentose phosphate pathway, galactose metabolism, and secondary metabolism. As such, Rdark was time-, type- (C3/C4) and species-dependent, due to the use of different substrates.

Reduced global plant respiration due to the acclimation of leaf dark respiration coupled with photosynthesis.

Leaf dark respiration (Rd ) acclimates to environmental changes. However, the magnitude, controls and time scales of acclimation remain unclear and are inconsistently treated in ecosystem models. We hypothesized that Rd and Rubisco carboxylation capacity (Vcmax ) at 25°C (Rd,25 , Vcmax,25 ) are coordinated so that Rd,25 variations support Vcmax,25 at a level allowing full light use, with Vcmax,25 reflecting daytime conditions (for photosynthesis), and Rd,25 /Vcmax,25 reflecting night-time conditions (for starch degradation and sucrose export). We tested this hypothesis temporally using a 5-yr warming experiment, and spatially using an extensive field-measurement data set. We compared the results to three published alternatives: Rd,25 declines linearly with daily average prior temperature; Rd at average prior night temperatures tends towards a constant value; and Rd,25 /Vcmax,25 is constant. Our hypothesis accounted for more variation in observed Rd,25 over time (R2 = 0.74) and space (R2 = 0.68) than the alternatives. Night-time temperature dominated the seasonal time-course of Rd , with an apparent response time scale of c. 2 wk. Vcmax dominated the spatial patterns. Our acclimation hypothesis results in a smaller increase in global Rd in response to rising CO2 and warming than is projected by the two of three alternative hypotheses, and by current models.

Photosynthesis in newly developed leaves of heat-tolerant wheat acclimates to long-term nocturnal warming.

We examined photosynthetic traits of pre-existing and newly developed flag leaves of four wheat genotypes grown in controlled-environment experiments. In newly developed leaves, acclimation of the maximum rate of net CO2 assimilation (An) to warm nights (i.e. increased An) was associated with increased capacity of Rubisco carboxylation and photosynthetic electron transport, with Rubisco activation state probably contributing to increased Rubisco activity. Metabolite profiling linked acclimation of An to greater accumulation of monosaccharides and saturated fatty acids in leaves; these changes suggest roles for osmotic adjustment of leaf turgor pressure and maintenance of cell membrane integrity. By contrast, where An decreased under warm nights, the decline was related to lower stomatal conductance and rates of photosynthetic electron transport. Decreases in An occurred despite higher basal PSII thermal stability in all genotypes exposed to warm nights: Tcrit of 45-46.5 °C in non-acclimated versus 43.8-45 °C in acclimated leaves. Pre-existing leaves showed no change in An-temperature response curves, except for an elite heat-tolerant genotype. These findings illustrate the impact of night-time warming on the ability of wheat plants to photosynthesize during the day, thereby contributing to explain the impact of global warming on crop productivity.

Heat tolerance of a tropical-subtropical rainforest tree species Polyscias elegans: time-dependent dynamic responses of physiological thermostability and biochemistry.

Heat stress interrupts physiological thermostability and triggers biochemical responses that are essential for plant survival. However, there is limited knowledge on the speed plants adjust to heat in hours and days, and which adjustments are crucial. Tropical-subtropical rainforest tree species (Polyscias elegans) were heated at 40°C for 5 d, before returning to 25°C for 13 d of recovery. Leaf heat tolerance was quantified using the temperature at which minimal chl a fluorescence sharply rose (Tcrit ). Tcrit , metabolites, heat shock protein (HSP) abundance and membrane lipid fatty acid (FA) composition were quantified. Tcrit increased by 4°C (48-52°C) within 2 h of 40°C exposure, along with rapid accumulation of metabolites and HSPs. By contrast, it took > 2 d for FA composition to change. At least 2 d were required for Tcrit , HSP90, HSP70 and FAs to return to prestress levels. The results highlight the multi-faceted response of P. elegans to heat stress, and how this response varies over the scale of hours to days, culminating in an increased level of photosynthetic heat tolerance. These responses are important for survival of plants when confronted with heat waves amidst ongoing global climate change.

Global mapping of RNA-chromatin contacts reveals a proximity-dominated connectivity model for ncRNA-gene interactions.

Non-coding RNAs (ncRNAs) are transcribed throughout the genome and provide regulatory inputs to gene expression through their interaction with chromatin. Yet, the genomic targets and functions of most ncRNAs are unknown. Here we use chromatin-associated RNA sequencing (ChAR-seq) to map the global network of ncRNA interactions with chromatin in human embryonic stem cells and the dynamic changes in interactions during differentiation into definitive endoderm. We uncover general principles governing the organization of the RNA-chromatin interactome, demonstrating that nearly all ncRNAs exclusively interact with genes in close three-dimensional proximity to their locus and provide a model predicting the interactome. We uncover RNAs that interact with many loci across the genome and unveil thousands of unannotated RNAs that dynamically interact with chromatin. By relating the dynamics of the interactome to changes in gene expression, we demonstrate that activation or repression of individual genes is unlikely to be controlled by a single ncRNA.

Rubisco deactivation and chloroplast electron transport rates co-limit photosynthesis above optimal leaf temperature in terrestrial plants.

Net photosynthetic CO2 assimilation rate (An) decreases at leaf temperatures above a relatively mild optimum (Topt) in most higher plants. This decline is often attributed to reduced CO2 conductance, increased CO2 loss from photorespiration and respiration, reduced chloroplast electron transport rate (J), or deactivation of Ribulose-1,5-bisphosphate Carboxylase Oxygenase (Rubisco). However, it is unclear which of these factors can best predict species independent declines in An at high temperature. We show that independent of species, and on a global scale, the observed decline in An with rising temperatures can be effectively accounted for by Rubisco deactivation and declines in J. Our finding that An declines with Rubisco deactivation and J supports a coordinated down-regulation of Rubisco and chloroplast electron transport rates to heat stress. We provide a model that, in the absence of CO2 supply limitations, can predict the response of photosynthesis to short-term increases in leaf temperature.

Coordination of photosynthetic traits across soil and climate gradients.

"Least-cost theory" posits that C3 plants should balance rates of photosynthetic water loss and carboxylation in relation to the relative acquisition and maintenance costs of resources required for these activities. Here we investigated the dependency of photosynthetic traits on climate and soil properties using a new Australia-wide trait dataset spanning 528 species from 67 sites. We tested the hypotheses that plants on relatively cold or dry sites, or on relatively more fertile sites, would typically operate at greater CO2 drawdown (lower ratio of leaf internal to ambient CO2 , Ci :Ca ) during light-saturated photosynthesis, and at higher leaf N per area (Narea ) and higher carboxylation capacity (Vcmax 25 ) for a given rate of stomatal conductance to water vapour, gsw . These results would be indicative of plants having relatively higher water costs than nutrient costs. In general, our hypotheses were supported. Soil total phosphorus (P) concentration and (more weakly) soil pH exerted positive effects on the Narea -gsw and Vcmax 25 -gsw slopes, and negative effects on Ci :Ca . The P effect strengthened when the effect of climate was removed via partial regression. We observed similar trends with increasing soil cation exchange capacity and clay content, which affect soil nutrient availability, and found that soil properties explained similar amounts of variation in the focal traits as climate did. Although climate typically explained more trait variation than soil did, together they explained up to 52% of variation in the slope relationships and soil properties explained up to 30% of the variation in individual traits. Soils influenced photosynthetic traits as well as their coordination. In particular, the influence of soil P likely reflects the Australia's geologically ancient low-relief landscapes with highly leached soils. Least-cost theory provides a valuable framework for understanding trade-offs between resource costs and use in plants, including limiting soil nutrients.

Enhancing crop yields through improvements in the efficiency of photosynthesis and respiration.

The rate with which crop yields per hectare increase each year is plateauing at the same time that human population growth and other factors increase food demand. Increasing yield potential ( Y p ) of crops is vital to address these challenges. In this review, we explore a component of Y p that has yet to be optimised - that being improvements in the efficiency with which light energy is converted into biomass ( ε c ) via modifications to CO2 fixed per unit quantum of light (α), efficiency of respiratory ATP production ( ε prod ) and efficiency of ATP use ( ε use ). For α, targets include changes in photoprotective machinery, ribulose bisphosphate carboxylase/oxygenase kinetics and photorespiratory pathways. There is also potential for ε prod to be increased via targeted changes to the expression of the alternative oxidase and mitochondrial uncoupling pathways. Similarly, there are possibilities to improve ε use via changes to the ATP costs of phloem loading, nutrient uptake, futile cycles and/or protein/membrane turnover. Recently developed high-throughput measurements of respiration can serve as a proxy for the cumulative energy cost of these processes. There are thus exciting opportunities to use our growing knowledge of factors influencing the efficiency of photosynthesis and respiration to create a step-change in yield potential of globally important crops.

The global spectrum of plant form and function: enhanced species-level trait dataset.

Here we provide the 'Global Spectrum of Plant Form and Function Dataset', containing species mean values for six vascular plant traits. Together, these traits -plant height, stem specific density, leaf area, leaf mass per area, leaf nitrogen content per dry mass, and diaspore (seed or spore) mass - define the primary axes of variation in plant form and function. The dataset is based on ca. 1 million trait records received via the TRY database (representing ca. 2,500 original publications) and additional unpublished data. It provides 92,159 species mean values for the six traits, covering 46,047 species. The data are complemented by higher-level taxonomic classification and six categorical traits (woodiness, growth form, succulence, adaptation to terrestrial or aquatic habitats, nutrition type and leaf type). Data quality management is based on a probabilistic approach combined with comprehensive validation against expert knowledge and external information. Intense data acquisition and thorough quality control produced the largest and, to our knowledge, most accurate compilation of empirically observed vascular plant species mean traits to date.

Molecular conflicts disrupting centromere maintenance contribute to Xenopus hybrid inviability.

Although central to evolution, the causes of hybrid inviability that drive reproductive isolation are poorly understood. Embryonic lethality occurs when the eggs of the frog X. tropicalis are fertilized with either X. laevis or X. borealis sperm. We observed that distinct subsets of paternal chromosomes failed to assemble functional centromeres, causing their mis-segregation during embryonic cell divisions. Core centromere DNA sequence analysis revealed little conservation among the three species, indicating that epigenetic mechanisms that normally operate to maintain centromere integrity are disrupted on specific paternal chromosomes in hybrids. In vitro reactions combining X. tropicalis egg extract with either X. laevis or X. borealis sperm chromosomes revealed that paternally matched or overexpressed centromeric histone CENP-A and its chaperone HJURP could rescue centromere assembly on affected chromosomes in interphase nuclei. However, although the X. laevis chromosomes maintained centromeric CENP-A in metaphase, X. borealis chromosomes did not and also displayed ultra-thin regions containing ribosomal DNA. Both centromere assembly and morphology of X. borealis mitotic chromosomes could be rescued by inhibiting RNA polymerase I or preventing the collapse of stalled DNA replication forks. These results indicate that specific paternal centromeres are inactivated in hybrids due to the disruption of associated chromatin regions that interfere with CENP-A incorporation, at least in some cases due to conflicts between replication and transcription machineries. Thus, our findings highlight the dynamic nature of centromere maintenance and its susceptibility to disruption in vertebrate interspecies hybrids.

Wheat photosystem II heat tolerance: evidence for genotype-by-environment interactions.

High temperature stress inhibits photosynthesis and threatens wheat production. One measure of photosynthetic heat tolerance is Tcrit - the critical temperature at which incipient damage to photosystem II (PSII) occurs. This trait could be improved in wheat by exploiting genetic variation and genotype-by-environment interactions (GEI). Flag leaf Tcrit of 54 wheat genotypes was evaluated in 12 thermal environments over 3 years in Australia, and analysed using linear mixed models to assess GEI effects. Nine of the 12 environments had significant genetic effects and highly variable broad-sense heritability (H2 ranged from 0.15 to 0.75). Tcrit GEI was variable, with 55.6% of the genetic variance across environments accounted for by the factor analytic model. Mean daily growth temperature in the month preceding anthesis was the most influential environmental driver of Tcrit GEI, suggesting biochemical, physiological and structural adjustments to temperature requiring different durations to manifest. These changes help protect or repair PSII upon exposure to heat stress, and may improve carbon assimilation under high temperature. To support breeding efforts to improve wheat performance under high temperature, we identified genotypes superior to commercial cultivars commonly grown by farmers, and demonstrated potential for developing genotypes with greater photosynthetic heat tolerance.

Wheat photosystem II heat tolerance responds dynamically to short- and long-term warming.

Wheat photosynthetic heat tolerance can be characterized using minimal chlorophyll fluorescence to quantify the critical temperature (Tcrit) above which incipient damage to the photosynthetic machinery occurs. We investigated intraspecies variation and plasticity of wheat Tcrit under elevated temperature in field and controlled-environment experiments, and assessed whether intraspecies variation mirrored interspecific patterns of global heat tolerance. In the field, wheat Tcrit varied diurnally-declining from noon through to sunrise-and increased with phenological development. Under controlled conditions, heat stress (36 °C) drove a rapid (within 2 h) rise in Tcrit that peaked after 3-4 d. The peak in Tcrit indicated an upper limit to PSII heat tolerance. A global dataset [comprising 183 Triticum and wild wheat (Aegilops) species] generated from the current study and a systematic literature review showed that wheat leaf Tcrit varied by up to 20 °C (roughly two-thirds of reported global plant interspecies variation). However, unlike global patterns of interspecies Tcrit variation that have been linked to latitude of genotype origin, intraspecific variation in wheat Tcrit was unrelated to that. Overall, the observed genotypic variation and plasticity of wheat Tcrit suggest that this trait could be useful in high-throughput phenotyping of wheat photosynthetic heat tolerance.

DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome wide.

Studies of genome regulation routinely use high-throughput DNA sequencing approaches to determine where specific proteins interact with DNA, and they rely on DNA amplification and short-read sequencing, limiting their quantitative application in complex genomic regions. To address these limitations, we developed directed methylation with long-read sequencing (DiMeLo-seq), which uses antibody-tethered enzymes to methylate DNA near a target protein's binding sites in situ. These exogenous methylation marks are then detected simultaneously with endogenous CpG methylation on unamplified DNA using long-read, single-molecule sequencing technologies. We optimized and benchmarked DiMeLo-seq by mapping chromatin-binding proteins and histone modifications across the human genome. Furthermore, we identified where centromere protein A localizes within highly repetitive regions that were unmappable with short sequencing reads, and we estimated the density of centromere protein A molecules along single chromatin fibers. DiMeLo-seq is a versatile method that provides multimodal, genome-wide information for investigating protein-DNA interactions.

Oxygen uptake rates have contrasting responses to temperature in the root meristem and elongation zone.

Growing at either 15 or 25°C, roots of Arabidopsis thaliana, Columbia accession, produce cells at the same rate and have growth zones of the same length. To determine whether this constancy is related to energetics, we measured oxygen uptake by means of a vibrating oxygen-selective electrode. Concomitantly, the spatial distribution of elongation was measured kinematically, delineating meristem and elongation zone. All seedlings were germinated, grown, and measured at a given temperature (15 or 25°C). Columbia was compared to lines where cell production rate roughly doubles between 15 and 25°C: Landsberg and two Columbia mutants, er-105 and ahk3-3. For all genotypes and temperatures, oxygen uptake rate at any position was highest at the root cap, where mitochondrial density was maximal, based on the fluorescence of a reporter. Uptake rate declined through the meristem to plateau within the elongation zone. For oxygen uptake rate integrated over a zone, the meristem had steady-state Q10 values ranging from 0.7 to 2.1; by contrast, the elongation zone had values ranging from 2.6 to 3.3, implying that this zone exerts a greater respiratory demand. These results highlight a substantial energy consumption by the root cap, perhaps helpful for maintaining hypoxia in stem cells, and suggest that rapid elongation is metabolically more costly than is cell division.

Dark respiration rates are not determined by differences in mitochondrial capacity, abundance and ultrastructure in C4 leaves.

Our understanding of the regulation of respiration in C4 plants, where mitochondria play different roles in the different types of C4 photosynthetic pathway, remains limited. We examined how leaf dark respiration rates (Rdark ), in the presence and absence of added malate, vary in monocots representing the three classical biochemical types of C4 photosynthesis (NADP-ME, NAD-ME and PCK) using intact leaves and extracted bundle sheath strands. In particular, we explored to what extent rates of Rdark are associated with mitochondrial number, volume and ultrastructure. Based on examination of a single species per C4 type, we found that the respiratory response of NAD-ME and PCK type bundle sheath strands to added malate was associated with differences in mitochondrial number, volume, and/or ultrastructure, while NADP-ME type bundle sheath strands did not respond to malate addition. In general, mitochondrial traits reflected the contributions mitochondria make to photosynthesis in the three C4 types. However, despite the obvious differences in mitochondrial traits, no clear correlation was observed between these traits and Rdark . We suggest that Rdark is primarily driven by cellular maintenance demands and not mitochondrial composition per se, in a manner that is somewhat independent of mitochondrial organic acid cycling in the light.

Barriers to initiating and maintaining participation in parkrun.

Interventions that increase population physical activity are required to promote health and wellbeing. parkrun delivers community-based, 5 km events worldwide yet 43% who register never participate in a parkrun event. This research had two objectives; i) explore the demographics of people who register for parkrun in United Kingdom, Australia, Ireland, and don't initiate or maintain participation ii) understand the barriers to participating in parkrun amongst these people. Mandatory data at parkrun registration provided demographic characteristics of parkrun registrants. A bespoke online survey distributed across the three countries captured the reasons for not participating or only participating once. Of 680,255 parkrun registrants between 2017 and 19, 293,542 (43%) did not participate in any parkrun events and 147,148 (22%) only participated in one parkrun event. Females, 16-34 years and physically inactive were more likely to not participate or not return to parkrun. Inconvenient start time was the most frequently reported barrier to participating, with females more likely than males to report the psychological barrier of feeling too unfit to participate. Co-creating strategies with and for people living with a chronic disease, women, young adults, and physically inactive people, could increase physical activity participation within parkrun.

Leaf nitrogen from the perspective of optimal plant function.

Leaf dry mass per unit area (LMA), carboxylation capacity (V cmax) and leaf nitrogen per unit area (Narea) and mass (Nmass) are key traits for plant functional ecology and ecosystem modelling. There is however no consensus about how these traits are regulated, or how they should be modelled. Here we confirm that observed leaf nitrogen across species and sites can be estimated well from observed LMA and V cmax at 25°C (V cmax25). We then test the hypothesis that global variations of both quantities depend on climate variables in specific ways that are predicted by leaf-level optimality theory, thus allowing both Narea to be predicted as functions of the growth environment.A new global compilation of field measurements was used to quantify the empirical relationships of leaf N to V cmax25 and LMA. Relationships of observed V cmax25 and LMA to climate variables were estimated, and compared to independent theoretical predictions of these relationships. Soil effects were assessed by analysing biases in the theoretical predictions.LMA was the most important predictor of Narea (increasing) and Nmass (decreasing). About 60% of global variation across species and sites in observed Narea, and 31% in Nmass, could be explained by observed LMA and V cmax25. These traits, in turn, were quantitatively related to climate variables, with significant partial relationships similar or indistinguishable from those predicted by optimality theory. Predicted trait values explained 21% of global variation in observed site-mean V cmax25, 43% in LMA and 31% in Narea. Predicted V cmax25 was biased low on clay-rich soils but predicted LMA was biased high, with compensating effects on Narea. Narea was overpredicted on organic soils. Synthesis. Global patterns of variation in observed site-mean Narea can be explained by climate-induced variations in optimal V cmax25 and LMA. Leaf nitrogen should accordingly be modelled as a consequence (not a cause) of V cmax25 and LMA, both being optimized to the environment. Nitrogen limitation of plant growth would then be modelled principally via whole-plant carbon allocation, rather than via leaf-level traits. Further research is required to better understand and model the terrestrial nitrogen and carbon cycles and their coupling.