A transcriptomic time-series reveals differing trajectories during pre-floral development in the apex and leaf in winter and spring varieties of Brassica napus.

Oilseed rape (Brassica napus) is an important global oil crop, with spring and winter varieties grown commercially. To understand the transcriptomic differences between these varieties, we collected transcriptomes from apex and leaf tissue from a spring variety, Westar, and a winter variety, Tapidor, before, during, and after vernalisation treatment, until the plants flowered. Large transcriptomic differences were noted in both varieties during the vernalisation treatment because of temperature and day length changes. Transcriptomic alignment revealed that the apex transcriptome reflects developmental state, whereas the leaf transcriptome is more closely aligned to the age of the plant. Similar numbers of copies of genes were expressed in both varieties during the time series, although key flowering time genes exhibited expression pattern differences. BnaFLC copies on A2 and A10 are the best candidates for the increased vernalisation requirement of Tapidor. Other BnaFLC copies show tissue-dependent reactivation of expression post-cold, with these dynamics suggesting some copies have retained or acquired a perennial nature. BnaSOC1 genes, also related to the vernalisation pathway, have expression profiles which suggest tissue subfunctionalisation. This understanding may help to breed varieties with more consistent or robust vernalisation responses, of special importance due to the milder winters resulting from climate change.

Significant Oxygen Underestimation When Quantifying Barium-Doped SrTiO Layers by Atom Probe Tomography.

In this paper, the capability for quantifying the composition of Ba-doped SrTiO layers from an atom probe measurement was explored. Rutherford backscattering spectrometry and time-of-flight/energy elastic recoil detection were used to benchmark the composition where the amount of titanium was intentionally varied between samples. The atom probe results showed a significant divergence from the benchmarked composition. The cause was shown to be a significant oxygen underestimation (≳14 at%). The ratio between oxygen and titanium for the samples varied between 2.6 and 12.7, while those measured by atom probe tomography were lower and covered a narrower range between 1.4 and 1.7. This difference was found to be associated with the oxygen and titanium predominantly field evaporating together as a molecular ion. The evaporation fields and bonding chemistries determined showed inconsistencies for explaining the oxygen underestimation and ion species measured. The measured ion charge state was in excellent agreement with that predicted by the Kingham postionization theory. Only by considering the measured ion species, their evaporation fields, the coordination chemistry, the analysis conditions, and some recently reported density functional theory modeling for oxide field emission were we able to postulate a field emission and oxygen neutral desorption process that may explain our results.

Assaying Effector Cell-to-Cell Mobility in Plant Tissues Identifies Hypermobility and Indirect Manipulation of Plasmodesmata.

In plants, plasmodesmata establish cytoplasmic continuity between cells to allow for communication and resource exchange across the cell wall. While plant pathogens use plasmodesmata as a pathway for both molecular and physical invasion, the benefits of molecular invasion (cell-to-cell movement of pathogen effectors) are poorly understood. To establish a methodology for identification and characterization of the cell-to-cell mobility of effectors, we performed a quantitative live imaging-based screen of candidate effectors of the fungal pathogen Colletotrichum higginsianum. We predicted C. higginsianum effectors by their expression profiles, the presence of a secretion signal, and their predicted and in planta localization when fused to green fluorescent protein. We assayed for cell-to-cell mobility of nucleocytosolic effectors and identified 14 that are cell-to-cell mobile. We identified that three of these effectors are "hypermobile," showing cell-to-cell mobility greater than expected for a protein of that size. To explore the mechanism of hypermobility, we chose two hypermobile effectors and measured their impact on plasmodesmata function and found that even though they show no direct association with plasmodesmata, each increases the transport capacity of plasmodesmata. Thus, our methods for quantitative analysis of cell-to-cell mobility of candidate microbe-derived effectors, or any suite of host proteins, can identify cell-to-cell hypermobility and offer greater understanding of how proteins affect plasmodesmal function and intercellular connectivity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

A causal inference and Bayesian optimisation framework for modelling multi-trait relationships-Proof-of-concept using Brassica napus seed yield under controlled conditions.

The improvement of crop yield is a major breeding target and there is a long history of research that has focussed on unravelling the mechanisms and processes that contribute to yield. Quantitative prediction of the interplay between morphological traits, and the effects of these trait-trait relationships on seed production remains, however, a challenge. Consequently, the extent to which crop varieties optimise their morphology for a given environment is largely unknown. This work presents a new combination of existing methodologies by framing crop breeding as an optimisation problem and evaluates the extent to which existing varieties exhibit optimal morphologies under the test conditions. In this proof-of-concept study using spring and winter oilseed rape plants grown under greenhouse conditions, we employ causal inference to model the hierarchically structured effects of 27 morphological yield traits on each other. We perform Bayesian optimisation of seed yield, to identify and quantify the morphologies of ideotype plants, which are expected to be higher yielding than the varieties in the studied panels. Under the tested growth conditions, we find that existing spring varieties occupy the optimal regions of trait-space, but that potentially high yielding strategies are unexplored in extant winter varieties. The same approach can be used to evaluate trait (morphology) space for any environment.

Delayed development of basal spikelets in wheat explains their increased floret abortion and rudimentary nature.

Large differences exist in the number of grains per spikelet across an individual wheat (Triticum aestivum L.) spike. The central spikelets produce the highest number of grains, while apical and basal spikelets are less productive, and the most basal spikelets are commonly only developed in rudimentary form. Basal spikelets are delayed in initiation, yet they continue to develop and produce florets. The precise timing or the cause of their abortion remains largely unknown. Here, we investigated the underlying causes of basal spikelet abortion using shading applications in the field. We found that basal spikelet abortion is likely to be the consequence of complete floret abortion, as both occur concurrently and have the same response to shading treatments. We detected no differences in assimilate availability across the spike. Instead, we show that the reduced developmental age of basal florets pre-anthesis is strongly associated with their increased abortion. Using the developmental age pre-abortion, we were able to predict final grain set per spikelet across the spike, alongside the characteristic gradient in the number of grains from basal to central spikelets. Future efforts to improve spikelet homogeneity across the spike could thus focus on improving basal spikelet establishment and increasing floret development rates pre-abortion.

Grasses exploit geometry to achieve improved guard cell dynamics.

Stomata are controllable micropores formed between two adjacent guard cells (GCs) that regulate gas flow across the plant surface.1 Grasses, among the most successful organisms on the planet and the main food crops for humanity, have GCs flanked by specialized lateral subsidiary cells (SCs).2,3,4 SCs improve performance by acting as a local pool of ions and metabolites to drive changes in turgor pressure within the GCs that open/close the stomatal pore.4,5,6,7,8 The 4-celled complex also involves distinctive changes in geometry, having dumbbell-shaped GCs compared with typical kidney-shaped stomata.2,4,9 However, the degree to which this distinctive geometry contributes to improved stomatal performance, and the underlying mechanism, remains unclear. To address this question, we created a finite element method (FEM) model of a grass stomatal complex that successfully captures experimentally observed pore opening/closure. Exploration of the model, including in silico and experimental mutant analyses, supports the importance of a reciprocal pressure system between GCs and SCs for effective stomatal function, with SCs functioning as springs to restrain lateral GC movement. Our results show that SCs are not essential but lead to a more responsive system. In addition, we show that GC wall anisotropy is not required for grass stomatal function (in contrast to kidney-shaped GCs10) but that a relatively thick GC rod region is needed to enhance pore opening. Our results demonstrate that a specific cellular geometry and associated mechanical properties are required for the effective functioning of grass stomata.

Reliability of the Critical Thinking in Communication Science and Disorders: A Content-Specific Critical Thinking Assessment.

INTRODUCTION: Students and professionals in communication sciences and disorders (CSD) need to exhibit good critical thinking (CT) skills when engaged in clinical tasks. CSD clinicians must make decisions that are free from biases and support their claim with facts. Thus, CSD clinicians need to be trained to question their clinical practices and to skeptically evaluate new practices that develop. A content-specific CT test can help determine if students are developing these skills. However, to date, no such content-specific CT assessment exists for CSD. The purpose of this study was to determine the reliability of the current version of a specific content CT assessment, the Critical Thinking in Communication Sciences and Disorders (CTCSD). METHODS: A sample of 150 CSD graduate students enrolled in three programs participated. They completed an online Qualtrics survey that consisted of the CTCSD. They completed the Qualtrics survey twice, once at the beginning of a semester and once at the end. The participant responses were independently scored by two research associates. The data were analyzed for reliability in three ways. Intra-subject reliability was assessed by comparing scores across the two testing sessions. Internal consistency of the items to measure a common construct was assessed using Cronbach's alpha and Guttman's Lambda 6. Inter-rater reliability was assessed using Cohen's Kappa coefficient. In addition, the time used to complete the survey was analyzed. RESULTS: The students from the three programs scored similarly on the CTCSD. High reliability ratings occurred for the intra-subject, internal consistency, and inter-rater measures. DISCUSSION/CONCLUSION: The results indicate the reliability of the CTCSD. In combination with previous results indicating the face, construct, and criterion validity of the CTCSD, it appears to have psychometric strength. The CTCSD may help academic and clinical faculty select learning activities and focus feedback to their graduate students in order to reinforce skills the students exhibit and to develop other skills.

Noncell-autonomous HSC70.1 chaperone displays homeostatic feedback regulation by binding its own mRNA.

The HSC70/HSP70 family of heat shock proteins are evolutionarily conserved chaperones involved in protein folding, protein transport, and RNA binding. Arabidopsis HSC70 chaperones are thought to act as housekeeping chaperones and as such are involved in many growth-related pathways. Whether Arabidopsis HSC70 binds RNA and whether this interaction is functional has remained an open question. We provide evidence that the HSC70.1 chaperone binds its own mRNA via its C-terminal short variable region (SVR) and inhibits its own translation. The SVR encoding mRNA region is necessary for HSC70.1 transcript mobility to distant tissues and that HSC70.1 transcript and not protein mobility is required to rescue root growth and flowering time of hsc70 mutants. We propose that this negative protein-transcript feedback loop may establish an on-demand chaperone pool that allows for a rapid response to stress. In summary, our data suggest that the Arabidopsis HSC70.1 chaperone can form a complex with its own transcript to regulate its translation and that both protein and transcript can act in a noncell-autonomous manner, potentially maintaining chaperone homeostasis between tissues.

Classification of wheat diseases using deep learning networks with field and glasshouse images.

Crop diseases can cause major yield losses, so the ability to detect and identify them in their early stages is important for disease control. Deep learning methods have shown promise in classifying multiple diseases; however, many studies do not use datasets that represent real field conditions, necessitating either further image processing or reducing their applicability. In this paper, we present a dataset of wheat images taken in real growth situations, including both field and glasshouse conditions, with five categories: healthy plants and four foliar diseases, yellow rust, brown rust, powdery mildew and Septoria leaf blotch. This dataset was used to train a deep learning model. The resulting model, named CerealConv, reached a 97.05% classification accuracy. When tested against trained pathologists on a subset of images from the larger dataset, the model delivered an accuracy score 2% higher than the best-performing pathologist. Image masks were used to show that the model was using the correct information to drive its classifications. These results show that deep learning networks are a viable tool for disease detection and classification in the field, and disease quantification is a logical next step.

Exact Bayesian inference for the detection of graft-mobile transcripts from sequencing data.

The long-distance transport of messenger RNAs (mRNAs) has been shown to be important for several developmental processes in plants. A popular method for identifying travelling mRNAs is to perform RNA-Seq on grafted plants. This approach depends on the ability to correctly assign sequenced mRNAs to the genetic background from which they originated. The assignment is often based on the identification of single-nucleotide polymorphisms (SNPs) between otherwise identical sequences. A major challenge is therefore to distinguish SNPs from sequencing errors. Here, we show how Bayes factors can be computed analytically using RNA-Seq data over all the SNPs in an mRNA. We used simulations to evaluate the performance of the proposed framework and demonstrate how Bayes factors accurately identify graft-mobile transcripts. The comparison with other detection methods using simulated data shows how not taking the variability in read depth, error rates and multiple SNPs per transcript into account can lead to incorrect classification. Our results suggest experimental design criteria for successful graft-mobile mRNA detection and show the pitfalls of filtering for sequencing errors or focusing on single SNPs within an mRNA.

Diffusion and bulk flow of amino acids mediate calcium waves in plants.

In plants, a variety of stimuli trigger long-range calcium signals that travel rapidly along the vasculature to distal tissues via poorly understood mechanisms. Here, we use quantitative imaging and analysis to demonstrate that traveling calcium waves are mediated by diffusion and bulk flow of amino acid chemical messengers. We propose that wounding triggers release of amino acids that diffuse locally through the apoplast, activating the calcium-permeable channel GLUTAMATE RECEPTOR-LIKE 3.3 as they pass. Over long distances through the vasculature, the wound-triggered dynamics of a fluorescent tracer show that calcium waves are likely driven by bulk flow of a channel-activating chemical. We observed that multiple stimuli trigger calcium waves with similar dynamics, but calcium waves alone cannot initiate all systemic defense responses, suggesting that mobile chemical messengers are a core component of complex systemic signaling in plants.

Intrinsically disordered plant protein PARCL colocalizes with RNA in phase-separated condensates whose formation can be regulated by mutating the PLD.

In higher plants, long-distance RNA transport via the phloem is crucial for communication between distant plant tissues to align development with stress responses and reproduction. Several recent studies suggest that specific RNAs are among the potential long-distance information transmitters. However, it is yet not well understood how these RNAs enter the phloem stream, how they are transported, and how they are released at their destination. It was proposed that phloem RNA-binding proteins facilitate RNA translocation. In the present study, we characterized two orthologs of the phloem-associated RNA chaperone-like (PARCL) protein from Arabidopsis thaliana and Brassica napus at functional and structural levels. Microscale thermophoresis showed that these phloem-abundant proteins can bind a broad spectrum of RNAs and show RNA chaperone activity in FRET-based in vitro assays. Our SAXS experiments revealed a high degree of disorder, typical for RNA-binding proteins. In agroinfiltrated tobacco plants, eYFP-PARCL proteins mainly accumulated in nuclei and nucleoli and formed cytosolic and nuclear condensates. We found that formation of these condensates was impaired by tyrosine-to-glutamate mutations in the predicted prion-like domain (PLD), while C-terminal serine-to-glutamate mutations did not affect condensation but reduced RNA binding and chaperone activity. Furthermore, our in vitro experiments confirmed phase separation of PARCL and colocalization of RNA with the condensates, while mutation as well as phosphorylation of the PLD reduced phase separation. Together, our results suggest that RNA binding and condensate formation of PARCL can be regulated independently by modification of the C-terminus and/or the PLD.

Field dependent study on the impact of co-evaporated multihits and ion pile-up for the apparent stoichiometric quantification of GaN and AlN.

For atom probe tomography, multihits and any associated ion pile-up are viewed as an "Achilles" heel when trying to establish accurate stoichiometric quantification. A significant reason for multihits and ion pile-up is credited to co-evaporation events. The impact is the underestimation of one or more elements present due to detector inadequacies when the field evaporated ions are spatially and temporally close. Nitride materials, especially GaN and AlN, have been shown to suffer a strong field dependent compositional bias, with N having the characteristics for being a species prone to ion pile-up. In this paper we have explored through field dependent measurements on GaN and AlN the associated impact of co-evaporated multihits and ion pile-up. To achieve this a normal CAMECA electrode along with a specially modified GRID electrode, which was designed to manipulate co-evaporated ions and hence ion pile-up, were employed. From our results and in-depth analysis, any co-evaporation and associated ion pile-up is found to be either very small, or not species dependent. Thus, ion pile-up cannot be attributed as the cause for the significant N underestimation observed in these materials.

Altering arabinans increases Arabidopsis guard cell flexibility and stomatal opening.

Stomata regulate plant water use and photosynthesis by controlling leaf gas exchange. They do this by reversibly opening the pore formed by two adjacent guard cells, with the limits of this movement ultimately set by the mechanical properties of the guard cell walls and surrounding epidermis.1,2 A body of evidence demonstrates that the methylation status and cellular patterning of pectin wall polymers play a core role in setting the guard cell mechanical properties, with disruption of the system leading to poorer stomatal performance.3-6 Here we present genetic and biochemical data showing that wall arabinans modulate guard cell flexibility and can be used to engineer stomata with improved performance. Specifically, we show that a short-chain linear arabinan epitope associated with the presence of rhamnogalacturonan I in the guard cell wall is required for full opening of the stomatal pore. Manipulations leading to the novel accumulation of longer-chain arabinan epitopes in guard cell walls led to an increase in the maximal pore aperture. Using computational modeling combined with atomic force microscopy, we show that this phenotype reflected a decrease in wall matrix stiffness and, consequently, increased flexing of the guard cells under turgor pressure, generating larger, rounder stomatal pores. Our results provide theoretical and experimental support for the conclusion that arabinan side chains of pectin modulate guard cell wall stiffness, setting the limits for cell flexing and, consequently, pore aperture, gas exchange, and photosynthetic assimilation.

High expression of the MADS-box gene VRT2 increases the number of rudimentary basal spikelets in wheat.

Spikelets are the fundamental building blocks of Poaceae inflorescences, and their development and branching patterns determine the various inflorescence architectures and grain yield of grasses. In wheat (Triticum aestivum), the central spikelets produce the most and largest grains, while spikelet size gradually decreases acropetally and basipetally, giving rise to the characteristic lanceolate shape of wheat spikes. The acropetal gradient corresponds with the developmental age of spikelets; however, the basal spikelets are developed first, and the cause of their small size and rudimentary development is unclear. Here, we adapted G&T-seq, a low-input transcriptomics approach, to characterize gene expression profiles within spatial sections of individual spikes before and after the establishment of the lanceolate shape. We observed larger differences in gene expression profiles between the apical, central, and basal sections of a single spike than between any section belonging to consecutive developmental time points. We found that SHORT VEGETATIVE PHASE MADS-box transcription factors, including VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT-A2), are expressed highest in the basal section of the wheat spike and display the opposite expression gradient to flowering E-class SEPALLATA1 genes. Based on multi-year field trials and transgenic lines, we show that higher expression of VRT-A2 in the basal sections of the spike is associated with increased numbers of rudimentary basal spikelets. Our results, supported by computational modeling, suggest that the delayed transition of basal spikelets from vegetative to floral developmental programs results in the lanceolate shape of wheat spikes. This study highlights the value of spatially resolved transcriptomics to gain insights into developmental genetics pathways of grass inflorescences.

An Investigation of Pulse Oximetry Levels During Swallowing in Healthy Adults and in Individuals with Severe and Very Severe Chronic Obstructive Pulmonary Disease.

To compare pulse oximetry (PO) levels during swallowing in healthy adults and adults with severe chronic obstructive pulmonary disease (COPD). Participants included two groups of 60 healthy adults, balanced for gender. The young group ranged from 18 to 38 years, and the older group from 60 to 87 years. In addition, there were 11 participants with COPD aged 43 to 82 years. PO levels were collected as each participant swallowed 10 mL of water, 10 mL of applesauce, and a piece of diced pear (three trials each). Analyses for the healthy groups revealed neither statistically significant main effects for bolus type nor interactions between gender and age. For between-subject effects, there was no main effect for gender, but there was a large main effect for age, and a gender and age interaction. In the group with COPD, there were no significant differences across bolus types; however, PO measures were consistently lower than the healthy groups for all bolus types. Healthy adults exhibited stable PO levels across bolus types. Adults with COPD, although exhibiting significantly lower PO levels, also remained stable. For clinicians who monitor PO measures, these results offer a more comprehensive understanding of the nature of these measures during swallowing in these groups.

Encoding, transmission, decoding, and specificity of calcium signals in plants.

Calcium acts as a signal and transmits information in all eukaryotes. Encoding machinery consisting of calcium channels, stores, buffers, and pumps can generate a variety of calcium transients in response to external stimuli, thus shaping the calcium signature. Mechanisms for the transmission of calcium signals have been described, and a large repertoire of calcium binding proteins exist that can decode calcium signatures into specific responses. Whilst straightforward as a concept, mysteries remain as to exactly how such information processing is biochemically implemented. Novel developments in imaging technology and genetically encoded sensors (such as calcium indicators), in particular for multi-signal detection, are delivering exciting new insights into intra- and intercellular calcium signaling. Here, we review recent advances in characterizing the encoding, transmission, and decoding mechanisms, with a focus on long-distance calcium signaling. We present technological advances and computational frameworks for studying the specificity of calcium signaling, highlight current gaps in our understanding and propose techniques and approaches for unravelling the underlying mechanisms.

Long-Distance Transported RNAs: From Identity to Function.

There is now a wealth of data, from different plants and labs and spanning more than two decades, which unequivocally demonstrates that RNAs can be transported over long distances, from the cell where they are transcribed to distal cells in other tissues. Different types of RNA molecules are transported, including micro- and messenger RNAs. Whether these RNAs are selected for transport and, if so, how they are selected and transported remain, in general, open questions. This aspect is likely not independent of the biological function and relevance of the transported RNAs, which are in most cases still unclear. In this review, we summarize the experimental data supporting selectivity or nonselectivity of RNA translocation and review the evidence for biological functions. After discussing potential issues regarding the comparability between experiments, we propose criteria that need to be critically evaluated to identify important signaling RNAs.

Aeciospore ejection in the rust pathogen Puccinia graminis is driven by moisture ingress.

Fungi have evolved an array of spore discharge and dispersal processes. Here, we developed a theoretical model that explains the ejection mechanics of aeciospore liberation in the stem rust pathogen Puccinia graminis. Aeciospores are released from cluster cups formed on its Berberis host, spreading early-season inoculum into neighboring small-grain crops. Our model illustrates that during dew or rainfall, changes in aeciospore turgidity exerts substantial force on neighboring aeciospores in cluster cups whilst gaps between spores become perfused with water. This perfusion coats aeciospores with a lubrication film that facilitates expulsion, with single aeciospores reaching speeds of 0.053 to 0.754 m·s-1. We also used aeciospore source strength estimates to simulate the aeciospore dispersal gradient and incorporated this into a publicly available web interface. This aids farmers and legislators to assess current local risk of dispersal and facilitates development of sophisticated epidemiological models to potentially curtail stem rust epidemics originating on Berberis.