Variation in Water-Holding Capacity in Sphagnum Species Depends on Both Plant and Colony Structure.

Peatlands have become a focal point in climate mitigation strategies as these ecosystems have significant carbon sequestration capacities when healthy but release CO2 and other greenhouse gases when damaged. However, as drought episodes become more frequent and prolonged, organisms key to the functioning of some peatlands are increasingly under pressure from desiccation. The Sphagnum mosses, which tend to keep their ecosystem waterlogged and many of whom promote peat formation, are only mildly desiccation-tolerant in comparison to other mosses. The role of Sphagnum anatomy and colony structure is poorly understood in the context of desiccation resilience. Using four different Sphagnum species belonging to four different subgenera and positions along the gradient of the water table, we show that plant morphological traits and colony density are important determinants of water storage capacity. Our results show that, as previously postulated, the majority of the water is stored in an easily exchangeable form, probably extracellularly, and that plant morphological traits, specifically the type and presence of branches, are major contributors to water storage and can explain some of the interspecies variation. We also show that plant density is another important determinant for water storage capacity as higher densities hold larger quantities of water per unit of biomass for all four species, which increases resilience to desiccation. The results presented here suggest that species choice and planting density should receive more attention when considering peatland restoration strategies.

Molecular and physiological responses to desiccation indicate the abscisic acid pathway is conserved in the peat moss, Sphagnum.

Mosses of the genus Sphagnum are the main components of peatlands, a major carbon-storing ecosystem. Changes in precipitation patterns are predicted to affect water relations in this ecosystem, but the effect of desiccation on the physiological and molecular processes in Sphagnum is still largely unexplored. Here we show that different Sphagnum species have differential physiological and molecular responses to desiccation but, surprisingly, this is not directly correlated with their position in relation to the water table. In addition, the expression of drought responsive genes is increased upon water withdrawal in all species. This increase in gene expression is accompanied by an increase in abscisic acid (ABA), supporting a role for ABA during desiccation responses in Sphagnum. Not only do ABA levels increase upon desiccation, but Sphagnum plants pre-treated with ABA display increased tolerance to desiccation, suggesting that ABA levels play a functional role in the response. In addition, many of the ABA signalling components are present in Sphagnum and we demonstrate, by complementation in Physcomitrium patens, that Sphagnum ABI3 is functionally conserved. The data presented here, therefore, support a conserved role for ABA in desiccation responses in Sphagnum.

Genetic architecture of variation in Arabidopsis thaliana rosettes.

Rosette morphology across Arabidopsis accessions exhibits considerable variation. Here we report a high-throughput phenotyping approach based on automatic image analysis to quantify rosette shape and dissect the underlying genetic architecture. Shape measurements of the rosettes in a core set of Recombinant Inbred Lines from an advanced mapping population (Multiparent Advanced Generation Inter-Cross or MAGIC) derived from inter-crossing 19 natural accessions. Image acquisition and analysis was scaled to extract geometric descriptors from time stamped images of growing rosettes. Shape analyses revealed heritable morphological variation at early juvenile stages and QTL mapping resulted in over 116 chromosomal regions associated with trait variation within the population. Many QTL linked to variation in shape were located near genes related to hormonal signalling and signal transduction pathways while others are involved in shade avoidance and transition to flowering. Our results suggest rosette shape arises from modular integration of sub-organ morphologies and can be considered a functional trait subjected to selective pressures of subsequent morphological traits. On an applied aspect, QTLs found will be candidates for further research on plant architecture.

Ectopic expression of Triticum polonicum VRT-A2 underlies elongated glumes and grains in hexaploid wheat in a dosage-dependent manner.

Flower development is an important determinant of grain yield in crops. In wheat (Triticum spp.), natural variation for the size of spikelet and floral organs is particularly evident in Triticum turgidum ssp. polonicum (also termed Triticum polonicum), a tetraploid subspecies of wheat with long glumes, lemmas, and grains. Using map-based cloning, we identified VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT2), which encodes a MADS-box transcription factor belonging to the SHORT VEGETATIVE PHASE family, as the gene underlying the T. polonicum long-glume (P1) locus. The causal P1 mutation is a sequence rearrangement in intron-1 that results in ectopic expression of the T. polonicum VRT-A2 allele. Based on allelic variation studies, we propose that the intron-1 mutation in VRT-A2 is the unique T. polonicum subspecies-defining polymorphism, which was later introduced into hexaploid wheat via natural hybridizations. Near-isogenic lines differing for the P1 locus revealed a gradient effect of P1 across spikelets and within florets. Transgenic lines of hexaploid wheat carrying the T. polonicum VRT-A2 allele show that expression levels of VRT-A2 are highly correlated with spike, glume, grain, and floral organ length. These results highlight how changes in expression profiles, through variation in cis-regulation, can affect agronomic traits in a dosage-dependent manner in polyploid crops.

A deep learning-integrated micro-CT image analysis pipeline for quantifying rice lodging resistance-related traits.

Lodging is a common problem in rice, reducing its yield and mechanical harvesting efficiency. Rice architecture is a key aspect of its domestication and a major factor that limits its high productivity. The ideal rice culm structure, including major_axis_culm, minor axis_culm, and wall thickness_culm, is critical for improving lodging resistance. However, the traditional method of measuring rice culms is destructive, time consuming, and labor intensive. In this study, we used a high-throughput micro-CT-RGB imaging system and deep learning (SegNet) to develop a high-throughput micro-CT image analysis pipeline that can extract 24 rice culm morphological traits and lodging resistance-related traits. When manual and automatic measurements were compared at the mature stage, the mean absolute percentage errors for major_axis_culm, minor_axis_culm, and wall_thickness_culm in 104 indica rice accessions were 6.03%, 5.60%, and 9.85%, respectively, and the R2 values were 0.799, 0.818, and 0.623. We also built models of bending stress using culm traits at the mature and tillering stages, and the R2 values were 0.722 and 0.544, respectively. The modeling results indicated that this method can quantify lodging resistance nondestructively, even at an early growth stage. In addition, we also evaluated the relationships of bending stress to shoot dry weight, culm density, and drought-related traits and found that plants with greater resistance to bending stress had slightly higher biomass, culm density, and culm area but poorer drought resistance. In conclusion, we developed a deep learning-integrated micro-CT image analysis pipeline to accurately quantify the phenotypic traits of rice culms in ∼4.6 min per plant; this pipeline will assist in future high-throughput screening of large rice populations for lodging resistance.

The Transcriptional Adaptor Protein ADA3a Modulates Flowering of Arabidopsis thaliana.

Histone acetylation is directly related to gene expression. In yeast, the acetyltransferase general control nonderepressible-5 (GCN5) targets histone H3 and associates with transcriptional co-activators alteration/deficiency in activation-2 (ADA2) and alteration/deficiency in activation-3 (ADA3) in complexes like SAGA. Arabidopsis thaliana has two genes encoding proteins, designated ADA3a and ADA3b, that correspond to yeast ADA3. We investigated the role of ADA3a and ADA3b in regulating gene expression during flowering time. Specifically, we found that knock out mutants ada3a-2 and the double mutant ada3a-2 ada3b-2 lead to early flowering compared to the wild type plants under long day (LD) conditions and after moving plants from short days to LD. Consistent with ADA3a being a repressor of floral initiation, FLOWERING LOCUS T (FT) expression was increased in ada3a mutants. In contrast, other genes involved in multiple pathways leading to floral transition, including FT repressors, players in GA signaling, and members of the SPL transcriptional factors, displayed reduced expression. Chromatin immunoprecipitation analysis revealed that ADA3a affects the histone H3K14 acetylation levels in SPL3, SPL5, RGA, GAI, and SMZ loci. In conclusion, ADA3a is involved in floral induction through a GCN5-containing complex that acetylates histone H3 in the chromatin of flowering related genes.

Deep Segmentation of Point Clouds of Wheat.

The 3D analysis of plants has become increasingly effective in modeling the relative structure of organs and other traits of interest. In this paper, we introduce a novel pattern-based deep neural network, Pattern-Net, for segmentation of point clouds of wheat. This study is the first to segment the point clouds of wheat into defined organs and to analyse their traits directly in 3D space. Point clouds have no regular grid and thus their segmentation is challenging. Pattern-Net creates a dynamic link among neighbors to seek stable patterns from a 3D point set across several levels of abstraction using the K-nearest neighbor algorithm. To this end, different layers are connected to each other to create complex patterns from the simple ones, strengthen dynamic link propagation, alleviate the vanishing-gradient problem, encourage link reuse and substantially reduce the number of parameters. The proposed deep network is capable of analysing and decomposing unstructured complex point clouds into semantically meaningful parts. Experiments on a wheat dataset verify the effectiveness of our approach for segmentation of wheat in 3D space.

Development of an Image Analysis Pipeline to Estimate Sphagnum Colony Density in the Field.

Sphagnum peatmosses play an important part in water table management of many peatland ecosystems. Keeping the ecosystem saturated, they slow the breakdown of organic matter and release of greenhouse gases, facilitating peatland's function as a carbon sink rather than a carbon source. Although peatland monitoring and restoration programs have increased recently, there are few tools to quantify traits that Sphagnum species display in their ecosystems. Colony density is often described as an important determinant in the establishment and performance in Sphagnum but detailed evidence for this is limited. In this study, we describe an image analysis pipeline that accurately annotates Sphagnum capitula and estimates plant density using open access computer vision packages. The pipeline was validated using images of different Sphagnum species growing in different habitats, taken on different days and with different smartphones. The developed pipeline achieves high accuracy scores, and we demonstrate its utility by estimating colony densities in the field and detecting intra and inter-specific colony densities and their relationship with habitat. This tool will enable ecologists and conservationists to rapidly acquire accurate estimates of Sphagnum density in the field without the need of specialised equipment.

Allotetraploidization in Brachypodium May Have Led to the Dominance of One Parent's Metabolome in Germinating Seeds.

Seed germination is a complex process during which a mature seed resumes metabolic activity to prepare for seedling growth. In this study, we performed a comparative metabolomic analysis of the embryo and endosperm using the community standard lines of three annual Brachypodium species, i.e., B. distachyon (Bd) and B. stacei (Bs) and their natural allotetraploid B. hybridum (BdBs) that has wider ecological range than the other two species. We explored how far the metabolomic impact of allotetraploidization would be observable as over-lapping changes at 4, 12, and 24 h after imbibition (HAI) with water when germination was initiated. Metabolic changes during germination were more prominent in Brachypodium embryos than in the endosperm. The embryo and endosperm metabolomes of Bs and BdBs were similar, and those of Bd were distinctive. The Bs and BdBs embryos showed increased levels of sugars and the tricarboxylic acid cycle compared to Bd, which could have been indicative of better nutrient mobilization from the endosperm. Bs and BdBs also showed higher oxalate levels that could aid nutrient transfer through altered cellular events. In Brachypodium endosperm, the thick cell wall, in addition to starch, has been suggested to be a source of nutrients to the embryo. Metabolites indicative of sugar metabolism in the endosperm of all three species were not prominent, suggesting that mobilization mostly occurred prior to 4 HAI. Hydroxycinnamic and monolignol changes in Bs and BdBs were consistent with cell wall remodeling that arose following the release of nutrients to the respective embryos. Amino acid changes in both the embryo and endosperm were broadly consistent across the species. Taking our data together, the formation of BdBs may have maintained much of the Bs metabolome in both the embryo and endosperm during the early stages of germination. In the embryo, this conserved Bs metabolome appeared to include an elevated sugar metabolism that played a vital role in germination. If these observations are confirmed in the future with more Brachypodium accessions, it would substantiate the dominance of the Bs metabolome in BdBs allotetraploidization and the use of metabolomics to suggest important adaptive changes.

Metabolomic Variation Aligns with Two Geographically Distinct Subpopulations of Brachypodium Distachyon before and after Drought Stress.

Brachypodium distachyon (Brachypodium) is a non-domesticated model grass that has been used to assess population level genomic variation. We have previously established a collection of 55 Brachypodium accessions that were sampled to reflect five different climatic regions of Turkey; designated 1a, 1c, 2, 3 and 4. Genomic and methylomic variation differentiated the collection into two subpopulations designated as coastal and central (respectively from regions 1a, 1c and the other from 2, 3 and 4) which were linked to environmental variables such as relative precipitation. Here, we assessed how far genomic variation would be reflected in the metabolomes and if this could be linked to an adaptive trait. Metabolites were extracted from eight-week-old seedlings from each accession and assessed using flow infusion high-resolution mass spectrometry (FIE-HRMS). Principal Component Analysis (PCA) of the derived metabolomes differentiated between samples from coastal and central subpopulations. The major sources of variation between seedling from the coastal and central subpopulations were identified. The central subpopulation was typified by significant increases in alanine, aspartate and glutamate metabolism and the tricarboxylic acid (TCA) cycle. Coastal subpopulation exhibited elevated levels of the auxin, indolacetic acid and rhamnose. The metabolomes of the seedling were also determined following the imposition of drought stress for seven days. The central subpopulation exhibited a metabolomic shift in response to drought, but no significant changes were seen in the coastal one. The drought responses in the central subpopulation were typified by changes in amino acids, increasing the glutamine that could be functioning as a stress signal. There were also changes in sugars that were likely to be an osmotic counter to drought, and changes in bioenergetic metabolism. These data indicate that genomic variation in our Turkish Brachypodium collection is largely reflected as distinctive metabolomes ("metabolotypes") through which drought tolerance might be mediated.

Interspecific and intraspecific phenotypic diversity for drought adaptation in bioenergy Arundo species.

Biomass crops are commonly grown in low-grade land and selection of drought-tolerant accessions is of major importance to sustain productivity. In this work, we assess phenotypic variation under different environmental scenarios in a series of accessions of Arundo donax, and contrast it with two closely related species, Arundo donaciformis and Arundo plinii. Gas-exchange and stomatal anatomy analysis showed an elevated photosynthetic capacity in A. plinii compared to A. donax and A. donaciformis with a significant intraspecific variation in A. donax. The three species showed significantly contrasting behaviour of transpiration under developing water stress and increasing vapour pressure deficit (VPD), with A. donax being the most conservative while A. plinii showed an elevated degree of insensitivity to environmental cues. Under optimal conditions, A. donax had the highest estimated leaf area (projected leaf area) and plant dry weight although a significant reduction under water stress was observed for A. donax and A. donaciformis accessions while no differences were recorded for A. plinii between optimal growing conditions (well-watered [WW]) and reduced soil water availability (water-stressed [WS]). A. donax displayed a markedly conservative water use behaviour but elevated sensitivity of biomass accumulation under stress conditions. By contrast, in A. plinii, biomass and transpiration were largely insensitive to WS and increasing VPD, though biomass dry weight under optimal conditions was significantly lower than A. donax. We provide evidence of interspecific phenotypic variation within the Arundo genus while the intraspecific phenotypic plasticity may be exploited for further selection of superior clones under disadvantageous environmental conditions. The extensive trade-off between water use and biomass accumulation present in the three species under stress conditions provides a series of novel traits to be exploited in the selection of superior clones adapted to different environmental scenarios. Non-destructive approaches are provided to screen large populations for water-stress-tolerant A. donax clones.

KnetMiner: a comprehensive approach for supporting evidence-based gene discovery and complex trait analysis across species.

The generation of new ideas and scientific hypotheses is often the result of extensive literature and database searches, but, with the growing wealth of public and private knowledge, the process of searching diverse and interconnected data to generate new insights into genes, gene networks, traits and diseases is becoming both more complex and more time-consuming. To guide this technically challenging data integration task and to make gene discovery and hypotheses generation easier for researchers, we have developed a comprehensive software package called KnetMiner which is open-source and containerized for easy use. KnetMiner is an integrated, intelligent, interactive gene and gene network discovery platform that supports scientists explore and understand the biological stories of complex traits and diseases across species. It features fast algorithms for generating rich interactive gene networks and prioritizing candidate genes based on knowledge mining approaches. KnetMiner is used in many plant science institutions and has been adopted by several plant breeding organizations to accelerate gene discovery. The software is generic and customizable and can therefore be readily applied to new species and data types; for example, it has been applied to pest insects and fungal pathogens; and most recently repurposed to support COVID-19 research. Here, we give an overview of the main approaches behind KnetMiner and we report plant-centric case studies for identifying genes, gene networks and trait relationships in Triticum aestivum (bread wheat), as well as, an evidence-based approach to rank candidate genes under a large Arabidopsis thaliana QTL. KnetMiner is available at: https://knetminer.org.

Total FLC transcript dynamics from divergent paralogue expression explains flowering diversity in Brassica napus.

Flowering time is a key adaptive and agronomic trait. In Arabidopsis, natural variation in expression levels of the floral repressor FLOWERING LOCUS C (FLC) leads to differences in vernalization. In Brassica napus there are nine copies of FLC. Here, we study how these multiple FLC paralogues determine vernalization requirement as a system. We collected transcriptome time series for Brassica napus spring, winter, semi-winter, and Siberian kale crop types. Modelling was used to link FLC expression dynamics to floral response following vernalization. We show that relaxed selection pressure has allowed expression of FLC paralogues to diverge, resulting in variation of FLC expression during cold treatment between paralogues and accessions. We find that total FLC expression dynamics best explains differences in cold requirement between cultivars, rather than expression of specific FLC paralogues. The combination of multiple FLC paralogues with different expression dynamics leads to rich behaviour in response to cold and a wide range of vernalization requirements in B. napus. We find evidence for different strategies to determine the response to cold in existing winter rapeseed accessions.

Semi-Automated Field Plot Segmentation From UAS Imagery for Experimental Agriculture.

We present an image processing method for accurately segmenting crop plots from Unmanned Aerial System imagery (UAS). The use of UAS for agricultural monitoring has increased significantly, emerging as a potentially cost effective alternative to manned aerial surveys and field work for remotely assessing crop state. The accurate segmentation of small densely-packed crop plots from UAS imagery over extensive areas is an important component of this monitoring activity in order to assess the state of different varieties and treatment regimes in a timely and cost-effective manner. Despite its importance, a reliable crop plot segmentation approach eludes us, with best efforts being relying on significant manual parameterization. The segmentation method developed uses a combination of edge detection and Hough line detection to establish the boundaries of each plot with pixel/point based metrics calculated for each plot segment. We show that with limited parameterization, segmentation of crop plots consistently over 89% accuracy are possible on different crop types and conditions. This is comparable to results obtained from rice paddies where the plant material in plots is sharply contrasted with the water, and represents a considerable improvement over previous methods for typical dry land crops.

A Functional Kinase Is Necessary for Cyclin-Dependent Kinase G1 (CDKG1) to Maintain Fertility at High Ambient Temperature in Arabidopsis.

Maintaining fertility in a fluctuating environment is key to the reproductive success of flowering plants. Meiosis and pollen formation are particularly sensitive to changes in growing conditions, especially temperature. We have previously identified cyclin-dependent kinase G1 (CDKG1) as a master regulator of temperature-dependent meiosis and this may involve the regulation of alternative splicing (AS), including of its own transcript. CDKG1 mRNA can undergo several AS events, potentially producing two protein variants: CDKG1L and CDKG1S, differing in their N-terminal domain which may be involved in co-factor interaction. In leaves, both isoforms have distinct temperature-dependent functions on target mRNA processing, but their role in pollen development is unknown. In the present study, we characterize the role of CDKG1L and CDKG1S in maintaining Arabidopsis fertility. We show that the long (L) form is necessary and sufficient to rescue the fertility defects of the cdkg1-1 mutant, while the short (S) form is unable to rescue fertility. On the other hand, an extra copy of CDKG1L reduces fertility. In addition, mutation of the ATP binding pocket of the kinase indicates that kinase activity is necessary for the function of CDKG1. Kinase mutants of CDKG1L and CDKG1S correctly localize to the cell nucleus and nucleus and cytoplasm, respectively, but are unable to rescue either the fertility or the splicing defects of the cdkg1-1 mutant. Furthermore, we show that there is partial functional overlap between CDKG1 and its paralog CDKG2 that could in part be explained by overlapping gene expression.

Genetic and Methylome Variation in Turkish Brachypodium Distachyon Accessions Differentiate Two Geographically Distinct Subpopulations.

Brachypodium distachyon (Brachypodium) is a non-domesticated model grass species that can be used to test if variation in genetic sequence or methylation are linked to environmental differences. To assess this, we collected seeds from 12 sites within five climatically distinct regions of Turkey. Seeds from each region were grown under standardized growth conditions in the UK to preserve methylated sequence variation. At six weeks following germination, leaves were sampled and assessed for genomic and DNA methylation variation. In a follow-up experiment, phenomic approaches were used to describe plant growth and drought responses. Genome sequencing and population structure analysis suggested three ancestral clusters across the Mediterranean, two of which were geographically separated in Turkey into coastal and central subpopulations. Phenotypic analyses showed that the coastal subpopulation tended to exhibit relatively delayed flowering and the central, increased drought tolerance as indicated by reduced yellowing. Genome-wide methylation analyses in GpC, CHG and CHH contexts also showed variation which aligned with the separation into coastal and central subpopulations. The climate niche modelling of both subpopulations showed a significant influence from the "Precipitation in the Driest Quarter" on the central subpopulation and "Temperature of the Coldest Month" on the coastal subpopulation. Our work demonstrates genetic diversity and variation in DNA methylation in Turkish accessions of Brachypodium that may be associated with climate variables and the molecular basis of which will feature in ongoing analyses.

Gradual polyploid genome evolution revealed by pan-genomic analysis of Brachypodium hybridum and its diploid progenitors.

Our understanding of polyploid genome evolution is constrained because we cannot know the exact founders of a particular polyploid. To differentiate between founder effects and post polyploidization evolution, we use a pan-genomic approach to study the allotetraploid Brachypodium hybridum and its diploid progenitors. Comparative analysis suggests that most B. hybridum whole gene presence/absence variation is part of the standing variation in its diploid progenitors. Analysis of nuclear single nucleotide variants, plastomes and k-mers associated with retrotransposons reveals two independent origins for B. hybridum, ~1.4 and ~0.14 million years ago. Examination of gene expression in the younger B. hybridum lineage reveals no bias in overall subgenome expression. Our results are consistent with a gradual accumulation of genomic changes after polyploidization and a lack of subgenome expression dominance. Significantly, if we did not use a pan-genomic approach, we would grossly overestimate the number of genomic changes attributable to post polyploidization evolution.

A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum.

The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.