Highlights from Faraday Discussion: Astrochemistry at high resolution, Baltimore, USA, May 2023.

The Faraday Discussion 'Astrochemistry at high resolution' was held at the Space Telescope Science Institute, Baltimore, United States, and online from May 31-June 2, 2023. The meeting brought together observers, modellers, and experimentalists at different career stages and from different countries to discuss advancements in astrochemistry resulting from improved spatial resolution, spectral resolution, and sensitivity. This conference report provides highlights of the meeting and summaries of the talks presented.

New interstellar laboratories in the molecular ring.

Much of what is known about chemistry in star-forming regions comes from observations of nearby (d < 500 pc) low-mass protostars. For chemistry in high-mass star-forming regions, several more distant (d ∼ 2-8 kpc), exceptionally bright molecular sources have also been the subjects of repeated observations but with concomitantly poorer linear spatial resolution. Facilities such as ALMA and JWST, however, now provide the means for observing distant sources at dramatically higher spatial resolution and sensitivity. We used the modest resolving power of the Atacama Compact Array, a dedicated subset of ALMA antennas, to carry out a pilot survey of 11 giant molecular clouds selected from the Bolocam Galactic Plane Survey [Battisti & Heyer, Astrophys. J., 2014, 780, 173] within the so-called molecular ring between about 4 and 8 kpc from the galactic center. Within our observed sample, molecular emission regions-most of which correspond to at least one (candidate) young stellar object-exhibit a range of chemical complexity and diversity. Furthermore, nine target giant molecular clouds contain well-fit methanol emission, giving us a first look at the spatial chemical variability within the objects at relatively high (compared to past observations) resolutions of ∼5''. This work lays the foundation for future high angular resolution studies of gas-phase chemistry with the full ALMA.

Spatially resolved gene regulatory networks in Asian rice (Oryza sativa cv. Nipponbare) leaves.

Transcriptome profiles in plants are heterogenous at every level of morphological organization. Even within organs, cells of the same type can have different patterns of gene expression depending on where they are positioned within tissues. This heterogeneity is associated with non-uniform distribution of biological processes within organs. The regulatory mechanisms that establish and sustain the spatial heterogeneity are unknown. Here, we identify regulatory modules that support functional specialization of different parts of Oryza sativa cv. Nipponbare leaves by leveraging transcriptome data, transcription factor binding motifs and global gene regulatory network prediction algorithms. We generated a global gene regulatory network in which we identified six regulatory modules that were active in different parts of the leaf. The regulatory modules were enriched for genes involved in spatially relevant biological processes, such as cell wall deposition, environmental sensing and photosynthesis. Strikingly, more than 86.9% of genes in the network were regulated by members of only five transcription factor families. We also generated targeted regulatory networks for the large MYB and bZIP/bHLH families to identify interactions that were masked in the global prediction. This analysis will provide a baseline for future single cell and array-based spatial transcriptome studies and for studying responses to environmental stress and demonstrates the extent to which seven coarse spatial transcriptome analysis can provide insight into the regulatory mechanisms supporting functional specialization within leaves.

Plant stem cells under low oxygen: metabolic rewiring by phytoglobin underlies stem cell functionality.

Root growth in maize (Zea mays L.) is regulated by the activity of the quiescent center (QC) stem cells located within the root apical meristem. Here, we show that despite being highly hypoxic under normal oxygen tension, QC stem cells are vulnerable to hypoxic stress, which causes their degradation with subsequent inhibition of root growth. Under low oxygen, QC stem cells became depleted of starch and soluble sugars and exhibited reliance on glycolytic fermentation with the impairment of the TCA cycle through the depressed activity of several enzymes, including pyruvate dehydrogenase (PDH). This finding suggests that carbohydrate delivery from the shoot might be insufficient to meet the metabolic demand of QC stem cells during stress. Some metabolic changes characteristic of the hypoxic response in mature root cells were not observed in the QC. Hypoxia-responsive genes, such as PYRUVATE DECARBOXYLASE (PDC) and ALCOHOL DEHYDROGENASE (ADH), were not activated in response to hypoxia, despite an increase in ADH activity. Increases in phosphoenolpyruvate (PEP) with little change in steady-state levels of succinate were also atypical responses to low-oxygen tensions. Overexpression of PHYTOGLOBIN 1 (ZmPgb1.1) preserved the functionality of the QC stem cells during stress. The QC stem cell preservation was underpinned by extensive metabolic rewiring centered around activation of the TCA cycle and retention of carbohydrate storage products, denoting a more efficient energy production and diminished demand for carbohydrates under conditions where nutrient transport may be limiting. Overall, this study provides an overview of metabolic responses occurring in plant stem cells during oxygen deficiency.

Growth-limiting drought stress induces time-of-day-dependent transcriptome and physiological responses in hybrid poplar.

Drought stress negatively impacts the health of long-lived trees. Understanding the genetic mechanisms that underpin response to drought stress is requisite for selecting or enhancing climate change resilience. We aimed to determine how hybrid poplars respond to prolonged and uniform exposure to drought; how responses to moderate and more severe growth-limiting drought stresses differed; and how drought responses change throughout the day. We established hybrid poplar trees (Populus × 'Okanese') from unrooted stem cutting with abundant soil moisture for 6 weeks. We then withheld water to establish well-watered, moderate and severe growth-limiting drought conditions. These conditions were maintained for 3 weeks during which growth was monitored. We then measured photosynthetic rates and transcriptomes of leaves that had developed during the drought treatments at two times of day. The moderate and severe drought treatments elicited distinct changes in growth and development, photosynthetic rates and global transcriptome profiles. Notably, the time of day of sampling produced the strongest effect in the transcriptome data. The moderate drought treatment elicited global transcriptome changes that were intermediate to the severe and well-watered treatments in the early evening but did not elicit a strong drought response in the morning. Stable drought conditions that are sufficient to limit plant growth elicit distinct transcriptional profiles depending on the degree of water limitation and on the time of day at which they are measured. There appears to be a limited number of genes and functional gene categories that are responsive to all of the tested drought conditions in this study emphasizing the complex nature of drought regulation in long-lived trees.

Host induced gene silencing of the Sclerotinia sclerotiorum ABHYDROLASE-3 gene reduces disease severity in Brassica napus.

Sclerotinia sclerotiorum is a pathogenic fungus that infects hundreds of crop species, causing extensive yield loss every year. Chemical fungicides are used to control this phytopathogen, but with concerns about increasing resistance and impacts on non-target species, there is a need to develop alternative control measures. In the present study, we engineered Brassica napus to constitutively express a hairpin (hp)RNA molecule to silence ABHYRDOLASE-3 in S. sclerotiorum. We demonstrate the potential for Host Induced Gene Silencing (HIGS) to protect B. napus from S. sclerotiorum using leaf, stem and whole plant infection assays. The interaction between the transgenic host plant and invading pathogen was further characterized at the molecular level using dual-RNA sequencing and at the anatomical level through microscopy to understand the processes and possible mechanisms leading to increased tolerance to this damaging necrotroph. We observed significant shifts in the expression of genes relating to plant defense as well as cellular differences in the form of structural barriers around the site of infection in the HIGS-protected plants. Our results provide proof-of-concept that HIGS is an effective means of limiting damage caused by S. sclerotiorum to the plant and demonstrates the utility of this biotechnology in the development of resistance against fungal pathogens.

Relationship between CH3OD Abundance and Temperature in the Orion KL Nebula.

The relative abundances of singly deuterated methanol isotopologues, [CH2DOH]/[CH3OD], in star-forming regions deviate from the statistically expected ratio of 3. In Orion KL, the nearest high-mass star-forming region to Earth, the singly deuterated methanol ratio is about 1, and the cause for this observation has been explored through theory for nearly three decades. We present high-angular resolution observations of Orion KL using the Atacama Large Millimeter/submillimeter Array to map small-scale changes in CH3OD column density across the nebula, which provide a new avenue to examine the deuterium chemistry during star and planet formation. By considering how CH3OD column densities vary with temperature, we find evidence of chemical processes that can significantly alter the observed gas-phase column densities. The astronomical data are compared with existing theoretical work and support D-H exchange between CH3OH and heavy water (i.e., HDO and D2O) at methanol's hydroxyl site in the icy mantles of dust grains. The enhanced CH3OD column densities are localized to the Hot Core-SW region, a pattern that may be linked to the coupled evolution of ice mantle chemistry and star formation in giant molecular clouds. This work provides new perspectives on deuterated methanol chemistry in Orion KL and informs considerations that may guide future theoretical, experimental, and observational work.

HIV-1 proviral landscape characterization varies by pipeline analysis.

INTRODUCTION: HIV rebounds after cessation of antiretroviral therapy, representing a barrier to cure. To better understand the virus reservoir, analysis pipelines have been developed that categorize proviral sequences as intact or defective, and further determine the precise nature of the sequence defects that may be present. We investigated the effects that different analysis pipelines had on the characterization of HIV-1 proviral sequences. METHODS: We used single genome amplification to generate near full-length (NFL) HIV-1 proviral DNA sequences, defined as amplicons greater than 8000 base pairs in length, isolated from peripheral blood mononuclear cells (PBMC) of treated suppressed participants with HIV-1. Amplicons underwent direct next-generation single genome sequencing and were analysed using four HIV-1 proviral characterization pipelines. Sequences were characterized as intact or defective; defective sequences were assessed for the number and types of defects present. To confirm and extend our findings, 691 proviruses from the Proviral Sequence Database (PSD) were analysed and the ProSeq-IT tool of the PSD was used to characterize both the participant and PSD proviruses. RESULTS AND DISCUSSION: Virus sequences derived from thirteen ART-treated virologically suppressed participants with HIV were studied. A total of 693 HIV-1 proviral sequences were generated, 282 of which were NFL. An average of 53 sequences per participant was analysed. We found that proviruses often harbour multiple sequence defect types (mean 2.7, 95% confidence interval [CI] 2.5, 3.0); the elimination order used by each pipeline affected the percentage of proviruses allotted into each defect category. These differences varied between participants, depending on the number of defect categories present in a given provirus sequence. Pipeline-specific differences in characterizing the HIV-1 5' untranslated region (5' UTR) led to an overestimation of the number of intact NFL proviral sequences, a finding corroborated in the independent PSD analysis. A comparison of the four published pipelines to ProSeq-IT found that ProSeq IT was more likely to characterize proviruses as intact. CONCLUSIONS: The choice of pipeline used for HIV-1 provirus landscape analysis may bias the classification of defective sequences. To improve the comparison of provirus characterizations across research groups, the development of a consensus elimination pipeline should be prioritized.

Single cell gene regulatory networks in plants: Opportunities for enhancing climate change stress resilience.

Global warming poses major challenges for plant survival and agricultural productivity. Thus, efforts to enhance stress resilience in plants are key strategies for protecting food security. Gene regulatory networks (GRNs) are a critical mechanism conferring stress resilience. Until recently, predicting GRNs of the individual cells that make up plants and other multicellular organisms was impeded by aggregate population scale measurements of transcriptome and other genome-scale features. With the advancement of high-throughput single cell RNA-seq and other single cell assays, learning GRNs for individual cells is now possible, in principle. In this article, we report on recent advances in experimental and analytical methodologies for single cell sequencing assays especially as they have been applied to the study of plants. We highlight recent advances and ongoing challenges for scGRN prediction, and finally, we highlight the opportunity to use scGRN discovery for studying and ultimately enhancing abiotic stress resilience in plants.

MET Exon 14-altered Lung Cancers and MET Inhibitor Resistance.

PURPOSE: MET tyrosine kinase inhibitors (TKIs) can achieve modest clinical outcomes in MET exon 14-altered lung cancers, likely secondary to primary resistance. Mechanisms of primary resistance remain poorly characterized and comprehensive proteomic analyses have not previously been performed. EXPERIMENTAL DESIGN: We performed hybrid capture-based DNA sequencing, targeted RNA sequencing, cell-free DNA sequencing, selected reaction monitoring mass spectrometry (SRM-MS), and immunohistochemistry on patient samples of MET exon 14-altered lung cancers treated with a MET TKI. Associations between overall response rate (ORR), progression-free survival (PFS), and putative genomic alterations and MET protein expression were evaluated. RESULTS: Seventy-five of 168 MET exon 14-altered lung cancers received a MET TKI. Previously undescribed (zygosity, clonality, whole-genome duplication) and known (copy-number focality, tumor mutational burden, mutation region/type) genomic factors were not associated with ORR/PFS (P > 0.05). In contrast, MET expression was associated with MET TKI benefit. Only cases with detectable MET expression by SRM-MS (N = 15) or immunochemistry (N = 22) responded to MET TKI therapy, and cancers with H-score ≥ 200 had a higher PFS than cancers below this cutoff (10.4 vs. 5.5 months, respectively; HR, 3.87; P = 0.02). CONCLUSIONS: In MET exon 14-altered cancers treated with a MET TKI, a comprehensive analysis of previously unknown and known genomic factors did not identify a genomic mechanism of primary resistance. Instead, MET expression correlated with benefit, suggesting the potential role of interrogating the proteome in addition to the genome in confirmatory prospective trials.

Thaumatin-Like Protein (TLP) Gene Family in Barley: Genome-Wide Exploration and Expression Analysis during Germination.

Thaumatin-like Proteins (TLPs) are known to play a vital role in plant defense, developmental processes and seed germination. We identified 19 TLP genes from the reference genome of barley and 37, 28 and 35 TLP genes from rice, Brachypodium and sorghum genomes, respectively. Comparative phylogenetic analysis classified the TLP family into nine groups. Localized gene duplications with diverse exon/intron structures contributed to the expansion of the TLP gene family in cereals. Most of the barley TLPs were localized on chromosome 5H. The spatiotemporal expression pattern of HvTLP genes indicated their predominant expression in the embryo, developing grains, root and shoot tissues. Differential expression of HvTLP14, HvTLP17 and HvTLP18 in the malting variety (Morex) over 16-96 h of grain germination revealed their possible role in malting. This study provides a description of the TLP gene family in barley and their possible involvement in seed germination and the malting process.

CNS Metastases in Patients With MET Exon 14-Altered Lung Cancers and Outcomes With Crizotinib.

PURPOSE: Although MET exon 14 (METex14)-altered lung cancers were first identified more than a decade and a half ago, the frequency of CNS metastatic disease remains poorly defined. Furthermore, the seminal trial of crizotinib in these patients (PROFILE 1001) did not report patterns of CNS response or progression. PATIENTS AND METHODS: Patients with pathologically confirmed, advanced non-small-cell lung cancers (NSCLC) harboring a METex14 alteration by targeted DNA/RNA sequencing were studied. The incidence of brain metastases and the outcomes of MET inhibition with crizotinib were analyzed. RESULTS: Eighty-three patients with METex14-altered metastatic NSCLC were identified. The incidence of CNS metastases at diagnosis was 17% (95% CI, 10% to 27%). The lifetime incidence was 36% (95% CI, 26% to 47%); 83% of patients had parenchymal disease, and 17% had leptomeningeal disease. The probability of having brain metastasis at 1, 2, and 3 years was 24%, 35%, and 38%, respectively. Fifty-four patients received crizotinib. The median time to radiologic CNS progression was 5.8 months (range, 3.7-20.0 months). Patterns of crizotinib progression were as follows: intracranial only in 10% of patients, intracranial and extracranial in 12%, and extracranial only in 78%. In patients with brain metastases before treatment, the median time on crizotinib was 7.5 months (range, 7.2-11.7 months). CONCLUSION: CNS metastases, including leptomeningeal disease, occurred in more than a third of patients with METex14-altered lung cancers. In crizotinib-treated patients with or without CNS metastases, CNS failure was seen in less than a quarter of patients on progression.

Genomic history and ecology of the geographic spread of rice.

Rice (Oryza sativa) is one of the world's most important food crops, and is comprised largely of japonica and indica subspecies. Here, we reconstruct the history of rice dispersal in Asia using whole-genome sequences of more than 1,400 landraces, coupled with geographic, environmental, archaeobotanical and paleoclimate data. Originating around 9,000 yr ago in the Yangtze Valley, rice diversified into temperate and tropical japonica rice during a global cooling event about 4,200 yr ago. Soon after, tropical japonica rice reached Southeast Asia, where it rapidly diversified, starting about 2,500 yr BP. The history of indica rice dispersal appears more complicated, moving into China around 2,000 yr BP. We also identify extrinsic factors that influence genome diversity, with temperature being a leading abiotic factor. Reconstructing the dispersal history of rice and its climatic correlates may help identify genetic adaptations associated with the spread of a key domesticated species.

Closing the Yield Gap for Cannabis: A Meta-Analysis of Factors Determining Cannabis Yield.

Until recently, the commercial production of Cannabis sativa was restricted to varieties that yielded high-quality fiber while producing low levels of the psychoactive cannabinoid tetrahydrocannabinol (THC). In the last few years, a number of jurisdictions have legalized the production of medical and/or recreational cannabis with higher levels of THC, and other jurisdictions seem poised to follow suit. Consequently, demand for industrial-scale production of high yield cannabis with consistent cannabinoid profiles is expected to increase. In this paper we highlight that currently, projected annual production of cannabis is based largely on facility size, not yield per square meter. This meta-analysis of cannabis yields reported in scientific literature aimed to identify the main factors contributing to cannabis yield per plant, per square meter, and per W of lighting electricity. In line with previous research we found that variety, plant density, light intensity and fertilization influence cannabis yield and cannabinoid content; we also identified pot size, light type and duration of the flowering period as predictors of yield and THC accumulation. We provide insight into the critical role of light intensity, quality, and photoperiod in determining cannabis yields, with particular focus on the potential for light-emitting diodes (LEDs) to improve growth and reduce energy requirements. We propose that the vast amount of genomics data currently available for cannabis can be used to better understand the effect of genotype on yield. Finally, we describe diversification that is likely to emerge in cannabis growing systems and examine the potential role of plant-growth promoting rhizobacteria (PGPR) for growth promotion, regulation of cannabinoid biosynthesis, and biocontrol.

Neural Net Classification Combined With Movement Analysis to Evaluate Setaria viridis as a Model System for Time of Day of Anther Appearance.

In many plant species, the time of day at which flowers open to permit pollination is tightly regulated. Proper time of flower opening, or Time of Day of Anther Appearance (TAA), may coordinate flowering opening with pollinator activity or may shift temperature sensitive developmental processes to cooler times of the day. The genetic mechanisms that regulate the timing of this process in cereal crops are unknown. To address this knowledge gap, it is necessary to establish a monocot model system that exhibits variation in TAA. Here, we examine the suitability of Setaria viridis, the model for C4 photosynthesis, for such a role. We developed an imaging system to monitor the temporal regulation of growth, flower opening time, and other physiological characteristics in Setaria. This system enabled us to compare Setaria varieties Ames 32254, Ames 32276, and PI 669942 variation in growth and daily flower opening time. We observed that TAA occurs primarily at night in these three Setaria accessions. However, significant variation between the accessions was observed for both the ratio of flowers that open in the day vs. night and the specific time of day where the rate is maximal. Characterizing this physiological variation is a requisite step toward uncovering the molecular mechanisms regulating TAA. Leveraging the regulation of TAA could provide researchers with a genetic tool to improve crop productivity in new environments.