Leaf thermotolerance of Hevea brasiliensis clones: intra- versus interclonal variation and relationships with other functional traits.

Land surface temperature is predicted to increase by 0.2 °C per decade due to climate change, although with considerable regional variability, and heatwaves are predicted to increase markedly in the future. These changes will affect where crops can be grown in the future. Understanding the thermal limits of plant physiological functioning and how flexible such limits are is thus important. Here, we report on the measurements of a core foliar thermotolerance trait, T50, defined as the temperature at which the maximum quantum yield (Fv/Fm) of photosystem II declines by 50%, across nine different Malaysian Hevea brasiliensis clones. We explore the relative importance of interclonal versus intraclonal variation in T50 as well as its association with leaf and hydraulic traits. We find very low variation in T50 within individual clones (mean intraclonal coefficient of variation (CoV) of 1.26%) and little variation across clones (interclonal CoV of 2.1%). The interclonal variation in T50 was lower than for all other functional traits considered. The T50 was negatively related to leaf mass per area and leaf dry matter content, but it was not related to hydraulic traits such as embolism resistance (P50) or hydraulic safety margins (HSM50). The range of T50 observed (42.9-46.2 °C) is well above the current maximum air temperatures Tmax,obs (T50 - Tmax,obs >5.8 °C), suggesting that H. brasiliensis is likely thermally safe in this south-east Asian region of Malaysia.

Decoding the gene regulatory network of endosperm differentiation in maize.

The persistent cereal endosperm constitutes the majority of the grain volume. Dissecting the gene regulatory network underlying cereal endosperm development will facilitate yield and quality improvement of cereal crops. Here, we use single-cell transcriptomics to analyze the developing maize (Zea mays) endosperm during cell differentiation. After obtaining transcriptomic data from 17,022 single cells, we identify 12 cell clusters corresponding to five endosperm cell types and revealing complex transcriptional heterogeneity. We delineate the temporal gene-expression pattern from 6 to 7 days after pollination. We profile the genomic DNA-binding sites of 161 transcription factors differentially expressed between cell clusters and constructed a gene regulatory network by combining the single-cell transcriptomic data with the direct DNA-binding profiles, identifying 181 regulons containing genes encoding transcription factors along with their high-confidence targets, Furthermore, we map the regulons to endosperm cell clusters, identify cell-cluster-specific essential regulators, and experimentally validated three predicted key regulators. This study provides a framework for understanding cereal endosperm development and function at single-cell resolution.

Beyond conflict: Kinship theory of intragenomic conflict predicts individual variation in altruistic behaviour.

Behavioural variation is essential for animals to adapt to different social and environmental conditions. The Kinship Theory of Intragenomic Conflict (KTIC) predicts that parent-specific alleles can support different behavioural strategies to maximize allele fitness. Previous studies, including in honey bees (Apis mellifera), supported predictions of the KTIC for parent-specific alleles to promote selfish behaviour. Here, we test the KTIC prediction that for altruism-promoting genes (i.e. those that promote behaviours that support the reproductive fitness of kin), the allele with the higher altruism optimum should be selected to be expressed while the other is silenced. In honey bee colonies, workers act altruistically when tending to the queen by performing a 'retinue' behaviour, distributing the queen's mandibular pheromone (QMP) throughout the hive. Workers exposed to QMP do not activate their ovaries, ensuring they care for the queen's brood instead of competing to lay unfertilized eggs. Due to the haplodiploid genetics of honey bees, the KTIC predicts that response to QMP is favoured by the maternal genome. We report evidence for parent-of-origin effects on the retinue response behaviour, ovarian development and gene expression in brains of worker honey bees exposed to QMP, consistent with the KTIC. Additionally, we show enrichment for genes with parent-of-origin expression bias within gene regulatory networks associated with variation in bees' response to QMP. Our study demonstrates that intragenomic conflict can shape diverse social behaviours and influence expression patterns of single genes as well as gene networks.

Tropical forest lianas have greater non-structural carbohydrate concentrations in the stem xylem than trees.

Lianas (woody vines) are important components of tropical forests and are known to compete with host trees for resources, decrease tree growth and increase tree mortality. Given the observed increases in liana abundance in some forests and their impacts on forest function, an integrated understanding of carbon dynamics of lianas and liana-infested host trees is critical for improved prediction of tropical forest responses to climate change. Non-structural carbohydrates (NSC) are the main substrate for plant metabolism (e.g., growth, respiration), and have been implicated in enabling tree survival under environmental stress, but little is known of how they vary among life-forms or of how liana infestation impacts host tree NSC. We quantified stem total NSC (NSC) concentrations and its fractions (starch and soluble sugars) in trees without liana infestation, trees with more than 50% of the canopy covered by lianas, and the lianas infesting those trees. We hypothesized that i) liana infestation depletes NSC storage in host trees by reducing carbon assimilation due to competition for resources; ii) trees and lianas, which greatly differ in functional traits related to water transport and carbon uptake, would also have large differences in NSC storage, and that As water availability has a significant role in NSC dynamics of Amazonian tree species, we tested these hypotheses within a moist site in western Amazonia and a drier forest site in southern Amazonia. We did not find any difference in NSC, starch or soluble sugar concentrations between infested and non-infested trees, in either site. This result suggests that negative liana impact on trees may be mediated through mechanisms other than depletion of host tree NSC concentrations. We found lianas have higher stem NSC and starch than trees in both sites. The consistent differences in starch concentrations, a long term NSC reserve, between life forms across sites reflect differences in carbon gain and use of lianas and trees. Soluble sugar concentrations were higher in lianas than in trees in the moist site but indistinguishable between life forms in the dry site. The lack of difference in soluble sugars between trees and lianas in the dry site emphasize the importance of this NSC fraction for plant metabolism of plants occurring in water limited environments. Abstract in Portuguese and Spanish are available in the supplementary material.

A maize epimerase modulates cell wall synthesis and glycosylation during stomatal morphogenesis.

The unique dumbbell-shape of grass guard cells (GCs) is controlled by their cell walls which enable their rapid responses to the environment. The molecular mechanisms regulating the synthesis and assembly of GC walls are as yet unknown. Here we have identified BZU3, a maize gene encoding UDP-glucose 4-epimerase that regulates the supply of UDP-glucose during GC wall synthesis. The BZU3 mutation leads to significant decreases in cellular UDP-glucose levels. Immunofluorescence intensities reporting levels of cellulose and mixed-linkage glucans are reduced in the GCs, resulting in impaired local wall thickening. BZU3 also catalyzes the epimerization of UDP-N-acetylgalactosamine to UDP-N-acetylglucosamine, and the BZU3 mutation affects N-glycosylation of proteins that may be involved in cell wall synthesis and signaling. Our results suggest that the spatiotemporal modulation of BZU3 plays a dual role in controlling cell wall synthesis and glycosylation via controlling UDP-glucose/N-acetylglucosamine homeostasis during stomatal morphogenesis. These findings provide insights into the mechanisms controlling formation of the unique morphology of grass stomata.

A defined roadmap of radial glia and astrocyte differentiation from human pluripotent stem cells.

Human gliogenesis remains poorly understood, and derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome. Here, we report controlled glial differentiation from hPSCs that bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro differentiation. hPSCs were first differentiated into radial glial cells (RGCs) resembling resident RGCs of the fetal telencephalon, and modulation of specific cell signaling pathways resulted in direct and stepwise induction of key astroglial markers (NFIA, NFIB, SOX9, CD44, S100B, glial fibrillary acidic protein [GFAP]). Transcriptomic and genome-wide epigenetic mapping and single-cell analysis confirmed RGC-to-astrocyte differentiation, obviating neurogenesis and the gliogenic switch. Detailed molecular and cellular characterization experiments uncovered new mechanisms and markers for human RGCs and astrocytes. In summary, establishment of a glia-exclusive neural lineage progression model serves as a unique serum-free platform of manufacturing large numbers of RGCs and astrocytes for neuroscience, disease modeling (e.g., Alexander disease), and regenerative medicine.

The Use of Flow Cytometry for Estimating Genome Sizes and DNA Ploidy Levels in Plants.

Flow cytometry has emerged as a uniquely flexible, accurate, and widely applicable technology for the analysis of plant cells. One of its most important applications centers on the measurement of nuclear DNA contents. This chapter describes the essential features of this measurement, outlining the overall methods and strategies, but going on to provide a wealth of technical details to ensure the most accurate and reproducible results. The chapter is aimed to be equally accessible to experienced plant cytometrists as well as those newly entering the field. Besides providing a step-by-step guide for estimating genome sizes and DNA-ploidy levels from fresh tissues, special attention is paid to the use of seeds and desiccated tissues for such purposes. Methodological aspects regarding field sampling, transport, and storage of plant material are also given in detail. Finally, troubleshooting information for the most common problems that may arise during the application of these methods is provided.

Differential gene expression underlying the biosynthesis of Dufour's gland signals in Bombus impatiens.

Pheromones regulating social behavior are one of the most explored phenomena in social insects. However, compound identity, biosynthesis and their genetic basis are known in only a handful of species. Here we examined the gene expression associated with pheromone biosynthesis of two main chemical classes: esters and terpenes, using the social bee Bombus impatiens. We conducted chemical and RNA-seq analyses of the Dufour's gland, an exocrine gland producing a plethora of pheromones regulating social behavior in hymenopteran species. The Dufour's gland contains mostly long-chained hydrocarbons, terpenes and esters that signal reproductive and social status in several bee species. In bumble bees, the Dufour's gland contains queen- and worker-specific esters, in addition to terpenes and terpene-esters only found in gynes and queens. These compounds are assumed to be synthesized de novo in the gland, however, their genetic basis is unknown. A whole transcriptome gene expression analysis of the gland in queens, gynes, queenless and queenright workers showed distinct transcriptomic profiles, with thousands of differentially expressed genes between the groups. Workers and queens express genes associated with key enzymes in the biosynthesis of wax esters, while queens and gynes preferentially express key genes in terpene biosynthesis. Overall, our data demonstrate gland-specific regulation of chemical signals associated with social behavior and identifies candidate genes and pathways regulating caste-specific chemical signals in social insects.

Anatomical functional traits and hydraulic vulnerability of trees in different water conditions in southern Amazonia.

PREMISE: Understanding tree species' responses to drought is critical for predicting the future of tropical forests, especially in regions where the climate is changing rapidly. METHODS: We compared anatomical and functional traits of the dominant tree species of two tropical forests in southern Amazonia, one on deep, well-drained soils (cerradão [CD]) and one in a riparian environment (gallery forest [GF]), to examine potential anatomical indicators of resistance or vulnerability to drought. RESULTS: Leaves of CD species generally had a thicker cuticle, upper epidermis, and mesophyll than those of GF species, traits that are indicative of adaptation to water deficit. In the GF, the theoretical hydraulic conductivity of the stems was significantly higher, indicating lower investment in drought resistance. The anatomical functional traits of CD species indicate a greater potential for surviving water restriction compared to the GF. Even so, it is possible that CD species could also be affected by extreme climate changes due to the more water-limited environment. CONCLUSIONS: In addition to the marked anatomical and functional differences between these phytophysiognomies, tree diversity within each is associated with a large range of hydraulic morphofunctional niches. Our results suggest the strong potential for floristic and functional compositional shifts under continued climate change, especially in the GF.

Long-term drought effects on the thermal sensitivity of Amazon forest trees.

The continued functioning of tropical forests under climate change depends on their resilience to drought and heat. However, there is little understanding of how tropical forests will respond to combinations of these stresses, and no field studies to date have explicitly evaluated whether sustained drought alters sensitivity to temperature. We measured the temperature response of net photosynthesis, foliar respiration and the maximum quantum efficiency of photosystem II (Fv /Fm ) of eight hyper-dominant Amazonian tree species at the world's longest-running tropical forest drought experiment, to investigate the effect of drought on forest thermal sensitivity. Despite a 0.6°C-2°C increase in canopy air temperatures following long-term drought, no change in overall thermal sensitivity of net photosynthesis or respiration was observed. However, photosystem II tolerance to extreme-heat damage (T50 ) was reduced from 50.0 ± 0.3°C to 48.5 ± 0.3°C under drought. Our results suggest that long-term reductions in precipitation, as projected across much of Amazonia by climate models, are unlikely to greatly alter the response of tropical forests to rising mean temperatures but may increase the risk of leaf thermal damage during heatwaves.

Circulating microRNA associated with future relapse status in major depressive disorder.

Major depressive disorder (MDD) is an episodic condition with relapsing and remitting disease course. Elucidating biomarkers that can predict future relapse in individuals responding to an antidepressant treatment holds the potential to identify those patients who are prone to illness recurrence. The current study explored relationships between relapse risk in recurrent MDD and circulating microRNAs (miRNAs) that participate in RNA silencing and post-transcriptional regulation of gene expression. Serum samples were acquired from individuals with a history of recurrent MDD who were followed longitudinally in the observational study, OBSERVEMDD0001 (ClinicalTrials.gov Identifier: NCT02489305). Circulating miRNA data were obtained in 63 participants who relapsed ("relapsers") and 154 participants who did not relapse ("non-relapsers") during follow-up. The miRNA was quantified using the ID3EAL™ miRNA Discovery Platform from MiRXES measuring 575 circulating miRNAs using a patented qPCR technology and normalized with a standard curve from spike-in controls in each plate. The association between miRNAs and subsequent relapse was tested using a linear model, adjusting for age, gender, and plate. Four miRNAs were nominally associated with relapse status during the observational follow-up phase with a false discover rate adjusted p-value < 0.1. Enrichment analysis of experimentally validated targets revealed 112 significantly enriched pathways, including neurogenesis, response to cytokine, neurotrophin signaling, vascular endothelial growth factor signaling, relaxin signaling, and cellular senescence pathways. These data suggest these miRNAs putatively associated with relapse status may have the potential to regulate genes involved in multiple signaling pathways that have previously been associated with MDD. If shown to be significant in a larger, independent sample, these data may hold potential for developing a miRNA signature to identify patients likely to relapse, allowing for earlier intervention.

A novel in situ passive heating method for evaluating whole-tree responses to daytime warming in remote environments.

BACKGROUND: Many significant ecosystems, including important non-forest woody ecosystems such as the Cerrado (Brazilian savannah), are under threat from climate change, yet our understanding of how increasing temperatures will impact native vegetation remains limited. Temperature manipulation experiments are important tools for investigating such impacts, but are often constrained by access to power supply and limited to low-stature species, juvenile individuals, or heating of target organs, perhaps not fully revealing how entire or mature individuals and ecosystems will react to higher temperatures. RESULTS: We present a novel, modified open top chamber design for in situ passive heating of whole individuals up to 2.5 m tall (but easily expandable) in remote field environments with strong solar irradiance. We built multiple whole-tree heating structures (WTHSs) in an area of Cerrado around native woody species Davilla elliptica and Erythroxylum suberosum to test the design and its effects on air temperature and humidity, while also studying the physiological responses of E. suberosum to short-term heating. The WTHSs raised internal air temperature by approximately 2.5 °C above ambient during the daytime. This increased to 3.4 °C between 09:00 and 17:00 local time when thermal impact was greatest, and during which time mean internal temperatures corresponded closely with maximum ambient temperatures. Heating was consistent over time and across WTHSs of variable size and shape, and they had minimal effect on humidity. E. suberosum showed no detectable response of photosynthesis or respiration to short-term experimental heating, but some indication of acclimation to natural temperature changes. CONCLUSIONS: Our WTHSs produced a consistent and reproducible level of daytime heating in line with mid-range climate predictions for the Cerrado biome by the end of the century. The whole-tree in situ passive heating design is flexible, low-cost, simple to build using commonly available materials, and minimises negative impacts associated with passive chambers. It could be employed to investigate the high temperature responses of many understudied species in a range of complex non-forest environments with sufficient solar irradiance, providing new and important insights into the possible impacts of our changing climate.

Relationships between species richness and ecosystem services in Amazonian forests strongly influenced by biogeographical strata and forest types.

Despite increasing attention for relationships between species richness and ecosystem services, for tropical forests such relationships are still under discussion. Contradicting relationships have been reported concerning carbon stock, while little is known about relationships concerning timber stock and the abundance of non-timber forest product producing plant species (NTFP abundance). Using 151 1-ha plots, we related tree and arborescent palm species richness to carbon stock, timber stock and NTFP abundance across the Guiana Shield, and using 283 1-ha plots, to carbon stock across all of Amazonia. We analysed how environmental heterogeneity influenced these relationships, assessing differences across and within multiple forest types, biogeographic regions and subregions. Species richness showed significant relationships with all three ecosystem services, but relationships differed between forest types and among biogeographical strata. We found that species richness was positively associated to carbon stock in all biogeographical strata. This association became obscured by variation across biogeographical regions at the scale of Amazonia, resembling a Simpson's paradox. By contrast, species richness was weakly or not significantly related to timber stock and NTFP abundance, suggesting that species richness is not a good predictor for these ecosystem services. Our findings illustrate the importance of environmental stratification in analysing biodiversity-ecosystem services relationships.

The effect of the brood and the queen on early gene expression in bumble bee workers' brains.

Worker reproduction in social insects is often regulated by the queen, but can be regulated by the brood and nestmates, who may use different mechanisms to induce the same outcomes in subordinates. Analysis of brain gene expression patterns in bumble bee workers (Bombus impatiens) in response to the presence of the queen, the brood, both or neither, identified 18 differentially expressed genes, 17 of them are regulated by the queen and none are regulated by the brood. Overall, brain gene expression differences in workers were driven by the queen's presence, despite recent studies showing that brood reduces worker egg laying and provides context to the queen pheromones. The queen affected important regulators of reproduction and brood care across insects, such as neuroparsin and vitellogenin, and a comparison with similar datasets in the honey bee and the clonal raider ant revealed that neuroparsin is differentially expressed in all species. These data emphasize the prominent role of the queen in regulating worker physiology and behavior. Genes that serve as key regulators of workers' reproduction are likely to play an important role in the evolution of sociality.

The maize single-nucleus transcriptome comprehensively describes signaling networks governing movement and development of grass stomata.

The unique morphology of grass stomata enables rapid responses to environmental changes. Deciphering the basis for these responses is critical for improving food security. We have developed a planta platform of single-nucleus RNA-sequencing by combined fluorescence-activated nuclei flow sorting, and used it to identify cell types in mature and developing stomata from 33,098 nuclei of the maize epidermis-enriched tissues. Guard cells (GCs) and subsidiary cells (SCs) displayed differential expression of genes, besides those encoding transporters, involved in the abscisic acid, CO2, Ca2+, starch metabolism, and blue light signaling pathways, implicating coordinated signal integration in speedy stomatal responses, and of genes affecting cell wall plasticity, implying a more sophisticated relationship between GCs and SCs in stomatal development and dumbbell-shaped guard cell formation. The trajectory of stomatal development identified in young tissues, and by comparison to the bulk RNA-seq data of the MUTE defective mutant in stomatal development, confirmed known features, and shed light on key participants in stomatal development. Our study provides a valuable, comprehensive, and fundamental foundation for further insights into grass stomatal function.

Chromosome-level Thlaspi arvense genome provides new tools for translational research and for a newly domesticated cash cover crop of the cooler climates.

Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field-based model species for genetics and epigenetics. The availability of a high-quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome-level genome assembly of var. MN106-Ref with improved gene annotation and use it to investigate gene structure differences between two accessions (MN108 and Spring32-10) that are highly amenable to genetic transformation. We describe non-coding RNAs, pseudogenes and transposable elements, and highlight tissue-specific expression and methylation patterns. Resequencing of forty wild accessions provided insights into genome-wide genetic variation, and QTL regions were identified for a seedling colour phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.

A Safety and Effectiveness Evaluation of a Callus Softener Containing Potassium Hydroxide.

BACKGROUND: Limited safety information has been described in the peer-reviewed literature for callus-softening products containing potassium hydroxide. METHODS: This pilot human use study evaluated the safety and effectiveness of a commercially available callus softener, containing less than 10% potassium hydroxide by weight. Baseline callused skin was scored (grade 1-4) on each study participant's feet (n = 10). Participants' feet were soaked and then a licensed manicurist applied a callus softener product to the right foot, which remained on callused skin for 3 to 5 minutes (no callus softener was applied to the participant's left foot). Both feet were then wiped with a wet towel, and a foot rasp was used to file the callused skin, beginning on the left foot. Callused skin was scored and participants' feet were evaluated by a physician immediately after use, 1 day after use, and 1 week after use for the presence or absence of skin irritation, adverse skin reactions, and chemical burns. RESULTS: No adverse events were reported by study participants or the physician for all evaluation time points. Each participant's highest callus grade score on the treated foot either improved or remained the same following product use (compared to baseline). Mean callus grade scores were 1.75 at baseline, 1.55 immediately after use, 1.25 1 day after use, and 1.50 1 week after use. CONCLUSIONS: Results from this pilot study suggest that callus-softening products containing less than 10% potassium hydroxide are likely to be safe and effective products under intended use scenarios of 3- to 5-minute application times, as dictated by product label instructions.

Fluorescence activated cell sorting-A selective tool for plant cell isolation and analysis.

Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single-cell suspensions. However, with few exceptions, higher plants comprise complex multicellular tissues and organs, in which the individual cells are held together by shared cell walls. Single-cell suspensions can be obtained through digestion of the cells walls and release of the so-called protoplasts (plants without their cell wall). Here we describe best practices for protoplast preparation, and for analysis through flow cytometry and cell sorting. Finally, the numerous downstream applications involving sorted protoplasts are discussed.

Application-based guidelines for best practices in plant flow cytometry.

Flow cytometry (FCM) is currently the most widely-used method to establish nuclear DNA content in plants. Since simple, 1-3-parameter, flow cytometers, which are sufficient for most plant applications, are commercially available at a reasonable price, the number of laboratories equipped with these instruments, and consequently new FCM users, has greatly increased over the last decade. This paper meets an urgent need for comprehensive recommendations for best practices in FCM for different plant science applications. We discuss advantages and limitations of establishing plant ploidy, genome size, DNA base composition, cell cycle activity, and level of endoreduplication. Applications of such measurements in plant systematics, ecology, molecular biology research, reproduction biology, tissue cultures, plant breeding, and seed sciences are described. Advice is included on how to obtain accurate and reliable results, as well as how to manage troubleshooting that may occur during sample preparation, cytometric measurements, and data handling. Each section is followed by best practice recommendations; tips as to what specific information should be provided in FCM papers are also provided.