Roadmap for the next decade of plant programmed cell death research.

Programmed cell death (PCD) is fundamentally important for plant development, abiotic stress responses and immunity, but our understanding of its regulation remains fragmented. Building a stronger research community is required to accelerate progress in this area through knowledge exchange and constructive debate. In this Viewpoint, we aim to initiate a collective effort to integrate data across a diverse set of experimental models to facilitate characterisation of the fundamental mechanisms underlying plant PCD and ultimately aid the development of a new plant cell death classification system in the future. We also put forward our vision for the next decade of plant PCD research stemming from discussions held during the 31st New Phytologist workshop, 'The Life and Death Decisions of Plant Cells' that took place at University College Dublin in Ireland (14-15 June 2023). We convey the key areas of significant progress and possible future research directions identified, including resolving the spatiotemporal control of cell death, isolation of its molecular and genetic regulators, and harnessing technical advances for studying PCD events in plants. Further, we review the breadth of potential impacts of plant PCD research and highlight the promising new applications of findings from this dynamically evolving field.

Gut microbiota influence anastomotic healing in colorectal cancer surgery through modulation of mucosal proinflammatory cytokines.

OBJECTIVE: Colorectal cancer (CRC) is the third most diagnosed cancer, and requires surgical resection and reconnection, or anastomosis, of the remaining bowel to re-establish intestinal continuity. Anastomotic leak (AL) is a major complication that increases mortality and cancer recurrence. Our objective is to assess the causal role of gut microbiota in anastomotic healing. DESIGN: The causal role of gut microbiota was assessed in a murine AL model receiving faecal microbiota transplantation (FMT) from patients with CRC collected before surgery and who later developed or not, AL. Anastomotic healing and gut barrier integrity were assessed after surgery. Bacterial candidates implicated in anastomotic healing were identified using 16S rRNA gene sequencing and were isolated from faecal samples to be tested both in vitro and in vivo. RESULTS: Mice receiving FMT from patients that developed AL displayed poor anastomotic healing. Profiling of gut microbiota of patients and mice after FMT revealed correlations between healing parameters and the relative abundance of Alistipes onderdonkii and Parabacteroides goldsteinii. Oral supplementation with A. onderdonkii resulted in a higher rate of leaks in mice, while gavage with P. goldsteinii improved healing by exerting an anti-inflammatory effect. Patients with AL and mice receiving FMT from AL patients presented upregulation of mucosal MIP-1α, MIP-2, MCP-1 and IL-17A/F before surgery. Retrospective analysis revealed that patients with AL present higher circulating neutrophil and monocyte counts before surgery. CONCLUSION: Gut microbiota plays an important role in surgical colonic healing in patients with CRC. The impact of these findings may extend to a vast array of invasive gastrointestinal procedures.

Microbial community origin and fate through a rural wastewater treatment plant.

Conventional wastewater treatment relies on a complex microbiota; however, much of this community is still to be characterized. To better understand the origin, dynamics and fate of bacteria within a wastewater treatment plant: untreated primary wastewater, activated sludge and post-treatment effluent were characterized. From 3163 exact sequence variants (ESVs), 860 were annotated to species-level. In primary wastewater, 28% of ESVs were putative bacterial species previously associated with humans, 14% with animals and 5% as common to the environment. Differential abundance analysis revealed significant relative reductions in ESVs from potentially human-associated species from primary wastewater to activated sludge, and significant increases in ESVs from species associated with nutrient cycling. Between primary wastewater and effluent, 51% of ESVs from human-associated species did not significantly differ, and species such as Bacteroides massiliensis and Bacteroides dorei increased. These findings illustrate that activated sludge increased extracellular protease and urease-producing species, ammonia and nitrite oxidizers, denitrifiers and specific phosphorus accumulators. Although many human-associated species declined, some persisted in effluent, including strains of potential health or environmental concern. Species-level microbial assessment may be useful for understanding variation in wastewater treatment efficiency as well as for monitoring the release of microbes into surface water and the wider ecosystem.

ANCHOR: a 16S rRNA gene amplicon pipeline for microbial analysis of multiple environmental samples.

Analysis of 16S ribosomal RNA (rRNA) gene amplification data for microbial barcoding can be inaccurate across complex environmental samples. A method, ANCHOR, is presented and designed for improved species-level microbial identification using paired-end sequences directly, multiple high-complexity samples and multiple reference databases. A standard operating procedure (SOP) is reported alongside benchmarking against artificial, single sample and replicated mock data sets. The method is then directly tested using a real-world data set from surface swabs of the International Space Station (ISS). Simple mock community analysis identified 100% of the expected species and 99% of expected gene copy variants (100% identical). A replicated mock community revealed similar or better numbers of expected species than MetaAmp, DADA2, Mothur and QIIME1. Analysis of the ISS microbiome identified 714 putative unique species/strains and differential abundance analysis distinguished significant differences between the Destiny module (U.S. laboratory) and Harmony module (sleeping quarters). Harmony was remarkably dominated by human gastrointestinal tract bacteria, similar to enclosed environments on earth; however, Destiny module bacteria also derived from nonhuman microbiome carriers present on the ISS, the laboratory's research animals. ANCHOR can help substantially improve sequence resolution of 16S rRNA gene amplification data within biologically replicated environmental experiments and integrated multidatabase annotation enhances interpretation of complex, nonreference microbiomes.

Comparative Transcriptomic Approaches Exploring Contamination Stress Tolerance in Salix sp. Reveal the Importance for a Metaorganismal de Novo Assembly Approach for Nonmodel Plants.

Metatranscriptomic study of nonmodel organisms requires strategies that retain the highly resolved genetic information generated from model organisms while allowing for identification of the unexpected. A real-world biological application of phytoremediation, the field growth of 10 Salix cultivars on polluted soils, was used as an exemplar nonmodel and multifaceted crop response well-disposed to the study of gene expression. Sequence reads were assembled de novo to create 10 independent transcriptomes, a global transcriptome, and were mapped against the Salix purpurea 94006 reference genome. Annotation of assembled contigs was performed without a priori assumption of the originating organism. Global transcriptome construction from 3.03 billion paired-end reads revealed 606,880 unique contigs annotated from 1588 species, often common in all 10 cultivars. Comparisons between transcriptomic and metatranscriptomic methodologies provide clear evidence that nonnative RNA can mistakenly map to reference genomes, especially to conserved regions of common housekeeping genes, such as actin, α/ß-tubulin, and elongation factor 1-α. In Salix, Rubisco activase transcripts were down-regulated in contaminated trees across all 10 cultivars, whereas thiamine thizole synthase and CP12, a Calvin Cycle master regulator, were uniformly up-regulated. De novo assembly approaches, with unconstrained annotation, can improve data quality; care should be taken when exploring such plant genetics to reduce de facto data exclusion by mapping to a single reference genome alone. Salix gene expression patterns strongly suggest cultivar-wide alteration of specific photosynthetic apparatus and protection of the antenna complexes from oxidation damage in contaminated trees, providing an insight into common stress tolerance strategies in a real-world phytoremediation system.

Meta-transcriptomics indicates biotic cross-tolerance in willow trees cultivated on petroleum hydrocarbon contaminated soil.

BACKGROUND: High concentrations of petroleum hydrocarbon (PHC) pollution can be hazardous to human health and leave soils incapable of supporting agricultural crops. A cheap solution, which can help restore biodiversity and bring land back to productivity, is cultivation of high biomass yielding willow trees. However, the genetic mechanisms which allow these fast-growing trees to tolerate PHCs are as yet unclear. METHODS: Salix purpurea 'Fish Creek' trees were pot-grown in soil from a former petroleum refinery, either lacking or enriched with C10-C50 PHCs. De novo assembled transcriptomes were compared between tree organs and impartially annotated without a priori constraint to any organism. RESULTS: Over 45% of differentially expressed genes originated from foreign organisms, the majority from the two-spotted spidermite, Tetranychus urticae. Over 99% of T. urticae transcripts were differentially expressed with greater abundance in non-contaminated trees. Plant transcripts involved in the polypropanoid pathway, including phenylalanine ammonia-lyase (PAL), had greater expression in contaminated trees whereas most resistance genes showed higher expression in non-contaminated trees. CONCLUSIONS: The impartial approach to annotation of the de novo transcriptomes, allowing for the possibility for multiple species identification, was essential for interpretation of the crop's response treatment. The meta-transcriptomic pattern of expression suggests a cross-tolerance mechanism whereby abiotic stress resistance systems provide improved biotic resistance. These findings highlight a valuable but complex biotic and abiotic stress response to real-world, multidimensional contamination which could, in part, help explain why crops such as willow can produce uniquely high biomass yields on challenging marginal land.

Dietary Intake Is Unlikely to Explain Symptom Severity and Syndrome-Specific Microbiome Alterations in a Cohort of Women with Fibromyalgia.

BACKGROUND: Significant alterations were recently identified in the composition and putative function of the gut microbiome in women with fibromyalgia. As diet can influence the composition of the gut microbiome, differences in nutritional intake could, in theory, account for some of these specific fibromyalgia microbiome alterations. The current study aims to compare the diet of women with fibromyalgia to that of controls in order to explore possible associations between the intake of certain nutrients, symptom severity and gut microbiome composition. METHODS: The study population was comprised of 56 women with fibromyalgia and 68 controls. Dietary intake was assessed using the NIH Automated Self-Administered 24 h recall, following dietitian's instructions and the completion of a three-day dietary recall. The gut microbiome was assessed by 16S ribosomal RNA gene sequencing of stool samples. RESULTS: Most demographic and anthropometric characteristics were comparable between groups. The average energy and macronutrient intake (total and relative) and overall diet quality score were not different between patients and controls, nor were the main vitamins, minerals, fatty acids, alcohol, caffeine, sugar or fiber intakes. The daily intake of micronutrients and normalized macronutrients in women with fibromyalgia was largely not correlated with disease-specific measures, including pain intensity, fatigue, cognitive symptoms and quality of sleep, or with the relative quantity of almost any of the gut microbiome bacterial taxa differentially abundant in fibromyalgia. CONCLUSION: These data demonstrate that dietary intakes, as evaluated by self-reported questionnaires, probably cannot explain the syndrome-specific differences in gut microbiome or the clinical phenotype of fibromyalgia.

Different Temperature and UV Patterns Modulate Berry Maturation and Volatile Compounds Accumulation in Vitis sp.

Volatile compounds (VCs) in grapevine berries play an important role in wine quality; however, such compounds and vine development can be sensitive to environmental conditions. Due to this sensitivity, changes in temperature patterns due to global warming are likely to further impact grape production and berry composition. The aim of this study was to determine the possible effects of different growing-degree day accumulation patterns on berry ripening and composition at harvest. An experimental field was conducted using Vitis sp. L'Acadie blanc, in Nova Scotia, Canada. Using on-the-row mini-greenhouses, moderate temperature increase and reduced ultraviolet (UV) exposure were triggered in grapevines during pre-veraison (inflorescence to the beginning of berry softening), post-veraison (berry softening to full maturity), and whole season (inflorescence to full maturity), while controls were left without treatment. Free and bound VCs were extracted from berries sampled at three different phenological stages between veraison and maturity before analysis by gas chromatography-mass spectrometry (GC-MS). Berries from grapevines exposed to higher temperatures during early berry development (pre-veraison and whole) accumulated significantly higher concentrations of benzene derivatives 2-phenylethanol and benzyl alcohol at harvest, but lower concentrations of hydroxy-methoxy-substituted volatile phenols, terpenes, and C13-norisoprenoids than the control berries. These results illustrate the importance of different environmental interactions in berry composition and suggest that temperature could potentially modulate phenylpropanoid and mevalonate metabolism in developing berries. This study provides insights into the relationships between abiotic conditions and secondary metabolism in grapevine and highlights the significance of early developmental stages on berry quality at harvest.

Emerging frontiers in human milk microbiome research and suggested primers for 16S rRNA gene analysis.

Human milk is the ideal food for infants due to its unique nutritional and immune properties, and more recently human milk has also been recognized as an important source of bacteria for infants. However, a substantial amount of fundamental human milk microbiome information remains unclear, such as the origin, composition and function of the community and its members. There is emerging evidence to suggest that the diversity and composition of the milk microbiome might differ between lactation stages, due to maternal factors and diet, agrarian and urban lifestyles, and geographical location. The evolution of the methods used for studying milk microbiota, transitioning from culture dependent-approaches to include culture-independent approaches, has had an impact on findings and, in particular, primer selection within 16S rRNA gene barcoding studies have led to discrepancies in observed microbiota communities. Here, the current state-of-the-art is reviewed and emerging frontiers essential to improving our understanding of the human milk microbiome are considered.

Distinct Changes Occur in the Human Breast Milk Microbiome Between Early and Established Lactation in Breastfeeding Guatemalan Mothers.

Human breast milk contains a diverse community of bacteria, but as breast milk microbiome studies have largely focused on mothers from high income countries where few women breastfeed to 6 months, the temporal changes in the breast milk microbiome that occur during later lactation stages have not been explored. For this cross-sectional study, microbiota from breast milk samples of Mam-Mayan mothers living in eight remote rural communities in the Western Highlands of Guatemala were analyzed. All mothers delivered vaginally and breastfed their infants for 6 months. Breast milk from 76 unrelated mothers was used to compare two lactation stages, either "early" (6-46 days post-partum, n = 33) or "late" (109-184 days post-partum, n = 43). Breast milk microbial communities were assessed using 16S ribosomal RNA gene sequencing and lactation stages were compared using DESeq2 differential abundance analysis. A total of 1,505 OTUs were identified, including 287 which could be annotated as putative species. Among several maternal factors, lactation stage explained microbiome variance and inertia in ordination with the most significance (p < 0.001). Differential abundance analysis identified 137 OTUs as significantly higher in either early or late lactation. These included a general shift from Staphylococcus and Streptococcus species in early lactation to Sphingobium and Pseudomonas species in late lactation. Species enriched in early lactation included putative commensal bacteria known to colonize the infant oral and intestinal tracts whereas species enriched in late lactation had a uniform functional trait associated with aromatic compound degradation. Differentially abundant species also included several species which have not previously been reported within breast milk, such as Janthinobacterium agaricidamnosum, Novosphingobium clariflavum, Ottowia beijingensis, and Flavobacterium cucumis. These discoveries describe temporal changes to the breast milk microbiome of healthy Guatemalan mothers from early to late lactation. Collectively, these findings illustrate how studying under-represented human populations might advance our understanding of factors that modulate the human milk microbiome in low and middle income countries (LMIC).

Altered microbiome composition in individuals with fibromyalgia.

Fibromyalgia (FM) is a prevalent syndrome, characterised by chronic widespread pain, fatigue, and impaired sleep, that is challenging to diagnose and difficult to treat. The microbiomes of 77 women with FM and that of 79 control participants were compared using 16S rRNA gene amplification and whole-genome sequencing. When comparing FM patients with unrelated controls using differential abundance analysis, significant differences were revealed in several bacterial taxa. Variance in the composition of the microbiomes was explained by FM-related variables more than by any other innate or environmental variable and correlated with clinical indices of FM. In line with observed alteration in butyrate-metabolising species, targeted serum metabolite analysis verified differences in the serum levels of butyrate and propionate in FM patients. Using machine-learning algorithms, the microbiome composition alone allowed for the classification of patients and controls (receiver operating characteristic area under the curve 87.8%). To the best of our knowledge, this is the first demonstration of gut microbiome alteration in nonvisceral pain. This observation paves the way for further studies, elucidating the pathophysiology of FM, developing diagnostic aids and possibly allowing for new treatment modalities to be explored.

Complementarity of three distinctive phytoremediation crops for multiple-trace element contaminated soil.

Trace element (TE) contaminated land represents an important risk to the environment and to human health worldwide. These soils usually contain a variety of TEs which can be a challenge for plant-based remediation options. As individual plant species often possess a limited range of TE remediation abilities, functional complementarity principles could be of value for remediation of soil contaminated by multiple TEs using assemblages of species. Monocultures and polycultures of Festuca arundinacea, Medicago sativa and Salix miyabeana were grown for 4months in aged-polluted soil contaminated by Ag, As, Cd, Cr, Cu, Pb, Se and Zn. Above and belowground biomass yields, root surface area (RSA) and TE tissue concentrations were recorded. In monoculture, the greatest aboveground biomass was produced by S. miyabeana (S), the greatest belowground biomass was from M. sativa (M) and F. arundinacea (F) produced the highest RSA. The polycultures of F+M, F+S and F+M+S produced among the highest values across all three traits. F. arundinacea monoculture and its combination with S. miyabeana (F+S) accumulated the highest amounts of total TEs in belowground tissues, whereas the most effective combination (or monoculture) for aboveground extraction yields varied depending on the TE considered. The crops demonstrated complementarity in their biomass allocation patterns as well as facilitative interactions. When considering contamination with a particular TE, the best phytomanagement approach could include a specific monoculture option; however, when above and belowground biomass allocation patterns, TE-remediation abilities as well as nitrogen accessibility are considered, co-cropping all three species (F+M+S) was the most robust scenario for remediation of multiple-TE contaminated land. By more effectively addressing a diversity of TE, species assemblage approaches could represent an important advancement towards enabling the use of plants to address contaminated-land issues worldwide.

The Response of a 16S Ribosomal RNA Gene Fragment Amplified Community to Lead, Zinc, and Copper Pollution in a Shanghai Field Trial.

Industrial and agricultural activities have caused extensive metal contamination of land throughout China and across the globe. The pervasive nature of metal pollution can be harmful to human health and can potentially cause substantial negative impact to the biosphere. To investigate the impact of anthropogenic metal pollution found in high concentrations in industrial, agricultural, and urban environments, 16S ribosomal RNA gene amplicon sequencing was used to track change in the amplified microbial community after metal contamination in a large-scale field experiment in Shanghai. A total of 1,566 operational taxonomic units (OTUs) identified from 448,108 sequences gathered from 20 plots treated as controls or with lead, zinc, copper, or all three metals. Constrained Analysis of Principal Coordinates ordination did not separate control and lead treatment but could separate control/lead, zinc, copper, and three metal treatment. DESeq2 was applied to identify 93 significantly differentially abundant OTUs varying in 211 pairwise instances between the treatments. Differentially abundant OTUs representing genera or species belonging to the phyla Chloroflexi, Cyanobacteria, Firmicutes, Latescibacteria, and Planctomycetes were almost universally reduced in abundance due to zinc, copper, or three metal treatment; with three metal treatment abolishing the detection of some OTUs, such as Leptolyngbya, Desmonostoc muscorum, and Microcoleus steenstrupii. The greatest increases due to metal treatment were observed in Bacteroidetes, Actinobacteria, Chlamydiae, Nitrospirae, and Proteobacteria (α, ß, δ, and γ); the most (relative) abundant being uncharacterized species within the genera Methylobacillus, Solirubrobacter, and Ohtaekwangia. Three metal treatment alone resulted in identification of 22 OTUs (genera or species) which were not detected in control soil, notably including Yonghaparkia alkaliphila, Pedobacter steynii, Pseudolabrys taiwanensis, Methylophilus methylotrophus, Nitrosospira, and Lysobacter mobilis. The capacity to track alterations of an amplified microbial community at high taxonomic resolution using modern bioinformatic approaches, as well as identifying where that resolution is lost for technical or biological reasons, provides an insight into the complexity of the microbial world resisting anthropogenic pollution. While functional assessment of uncharacterized organisms within environmental samples is technically challenging, an important step is observing those organisms able to tolerate extreme stress and to recognize the extent to which important amplifiable community members still require characterization.

Transcriptomic Response of Purple Willow (Salix purpurea) to Arsenic Stress.

Arsenic (As) is a toxic element for plants and one of the most common anthropogenic pollutants found at contaminated sites. Despite its severe effects on plant metabolism, several species can accumulate substantial amounts of arsenic and endure the associated stress. However, the genetic mechanisms involved in arsenic tolerance remains obscure in many model plant species used for land decontamination (phytoremediation), including willows. The present study assesses the potential of Salix purpurea cv. 'Fish Creek' for arsenic phytoextraction and reveals the genetic responses behind arsenic tolerance, phytoextraction and metabolism. Four weeks of hydroponic exposure to 0, 5, 30 and 100 mg/L revealed that plants were able to tolerate up to 5 mg/L arsenic. Concentrations of 0 and 5 mg/L of arsenic treatment were then used to compare alterations in gene expression of roots, stems and leaves using RNA sequencing. Differential gene expression revealed transcripts encoding proteins putatively involved in entry of arsenic into the roots, storage in vacuoles and potential transport through the plant as well as primary and secondary (indirect) toxicity tolerance mechanisms. A major role for tannin as a compound used to relieve cellular toxicity is implicated as well as unexpected expression of the cadmium transporter CAX2, providing a potential means for internal arsenic mobility. These insights into the underpinning genetics of a successful phytoremediating species present novel opportunities for selection of dedicated arsenic tolerant crops as well as the potential to integrate such tolerances into a wider Salix ideotype alongside traits including biomass yield, biomass quality, low agricultural inputs and phytochemical production.

Five willow varieties cultivated across diverse field environments reveal stem density variation associated with high tension wood abundance.

Sustainable and inexpensive production of biomass is necessary to make biofuel production feasible, but represents a challenge. Five short rotation coppice willow cultivars, selected for high biomass yield, were cultivated on sites at four diverse regions of Quebec in contrasting environments. Wood composition and anatomical traits were characterized. Tree height and stem diameter were measured to evaluate growth performance of the cultivars according to the diverse pedoclimatic conditions. Each cultivar showed very specific responses to its environment. While no significant variation in lignin content was observed between sites, there was variation between cultivars. Surprisingly, the pattern of substantial genotype variability in stem density was maintained across all sites. However, wood anatomy did differ between sites in a cultivar (producing high and low density wood), suggesting a probable response to an abiotic stress. Furthermore, twice as many cellulose-rich G-fibers, comprising over 50% of secondary xylem, were also found in the high density wood, a finding with potential to bring higher value to the lignocellulosic bioethanol industry.

Insights into nitrogen allocation and recycling from nitrogen elemental analysis and 15N isotope labelling in 14 genotypes of willow.

Minimizing nitrogen (N) fertilization inputs during cultivation is essential for sustainable production of bioenergy and biofuels. The biomass crop willow (Salix spp.) is considered to have low N fertilizer requirements due to efficient recycling of nutrients during the perennial cycle. To investigate how successfully different willow genotypes assimilate and allocate N during growth, and remobilize and consequently recycle N before the onset of winter dormancy, N allocation and N remobilization (to and between different organs) were examined in 14 genotypes of a genetic family using elemental analysis and (15)N as a label. Cuttings were established in pots in April and sampled in June, August and at onset of senescence in October. Biomass yield of the trees correlated well with yields recorded in the field. Genotype-specific variation was observed for all traits measured and general trends spanning these sampling points were identified when trees were grouped by biomass yield. Nitrogen reserves in the cutting fuelled the entirety of the canopy establishment, yet earlier cessation of this dependency was linked to higher biomass yields. The stem was found to be the major N reserve by autumn, which constitutes a major source of N loss at harvest, typically every 2-3 years. These data contribute to understanding N remobilization in short rotation coppice willow and to the identification of traits that could potentially be selected for in breeding programmes to further improve the sustainability of biomass production.