Phylotranscriptomics provides a treasure trove of flood-tolerance mechanisms in the Cardamineae tribe.

Flooding events are highly detrimental to most terrestrial plant species. However, there is an impressive diversity of plant species that thrive in flood-prone regions and represent a treasure trove of unexplored flood-resilience mechanisms. Here we surveyed a panel of four species from the Cardamineae tribe representing a broad tolerance range. This included the flood-tolerant Cardamine pratensis, Rorippa sylvestris and Rorippa palustris and the flood-sensitive species Cardamine hirsuta. All four species displayed a quiescent strategy, evidenced by the repression of shoot growth underwater. Comparative transcriptomics analyses between the four species and the sensitive model species Arabidopsis thaliana were facilitated via de novo transcriptome assembly and identification of 16 902 universal orthogroups at a high resolution. Our results suggest that tolerance likely evolved separately in the Cardamine and Rorippa species. While the Rorippa response was marked by a strong downregulation of cell-cycle genes, Cardamine minimized overall transcriptional regulation. However, a weak starvation response was a universal trait of tolerant species, potentially achieved in multiple ways. It could result from a strong decline in cell-cycle activity, but is also intertwined with autophagy, senescence, day-time photosynthesis and night-time fermentation capacity. Our data set provides a rich source to study adaptational mechanisms of flooding tolerance.

A tool for rapid, automated characterization of population epigenomics in plants.

Epigenetic variation in plant populations is an important factor in determining phenotype and adaptation to the environment. However, while advances have been made in the molecular and computational methods to analyze the methylation status of a given sample of DNA, tools to profile and compare the methylomes of multiple individual plants or groups of plants at high resolution and low cost are lacking. Here, we describe a computational approach and R package (sounDMR) that leverages the benefits of long read nanopore sequencing to enable robust identification of differential methylation from complex experimental designs, as well as assess the variability within treatment groups and identify individual plants of interest. We demonstrate the utility of this approach by profiling a population of Arabidopsis thaliana exposed to a demethylating agent and identify genomic regions of high epigenetic variability between individuals. Given the low cost of nanopore sequencing devices and the ease of sample preparation, these results show that high resolution epigenetic profiling of plant populations can be made more broadly accessible in plant breeding and biotechnology.

Keeping the shoot above water - submergence triggers antithetical growth responses in stems and petioles of watercress (Nasturtium officinale).

The molecular mechanisms controlling underwater elongation are based extensively on studies on internode elongation in the monocot rice (Oryza sativa) and petiole elongation in Rumex rosette species. Here, we characterize underwater growth in the dicot Nasturtium officinale (watercress), a wild species of the Brassicaceae family, in which submergence enhances stem elongation and suppresses petiole growth. We used a genome-wide transcriptome analysis to identify the molecular mechanisms underlying the observed antithetical growth responses. Though submergence caused a substantial reconfiguration of the petiole and stem transcriptome, only little qualitative differences were observed between both tissues. A core submergence response included hormonal regulation and metabolic readjustment for energy conservation, whereas tissue-specific responses were associated with defense, photosynthesis, and cell wall polysaccharides. Transcriptomic and physiological characterization suggested that the established ethylene, abscisic acid (ABA), and GA growth regulatory module for underwater elongation could not fully explain underwater growth in watercress. Petiole growth suppression is likely attributed to a cell cycle arrest. Underwater stem elongation is driven by an early decline in ABA and is not primarily mediated by ethylene or GA. An enhanced stem elongation observed in the night period was not linked to hypoxia and suggests an involvement of circadian regulation.

Climate explains population divergence in drought-induced plasticity of functional traits and gene expression in a South African Protea.

Long-term environmental variation often drives local adaptation and leads to trait differentiation across populations. Additionally, when traits change in an environment-dependent way through phenotypic plasticity, the genetic variation underlying plasticity will also be under selection. These processes could create a landscape of differentiation across populations in traits and their plasticity. Here, we performed a dry-down experiment under controlled conditions to measure responses in seedlings of a shrub species from the Cape Floristic Region, the common sugarbush (Protea repens). We measured morphological and physiological traits, and sequenced whole transcriptomes of leaf tissues from eight populations that represent both the climatic and the geographical distribution of this species. We found that there is substantial variation in how populations respond to drought, but we also observed common patterns such as reduced leaf size and leaf thickness, and up-regulation of stress-related and down-regulation of growth-related gene groups. Both high environmental heterogeneity and milder source site climates were associated with higher plasticity in various traits and co-expression gene networks. Associations between traits, trait plasticity, gene networks and the source site climate suggest that temperature may play a greater role in shaping these patterns when compared to precipitation, in line with recent changes in the region due to climate change. We also found that traits respond to climatic variation in an environment-dependent manner: some associations between traits and climate were apparent only under certain growing conditions. Together, our results uncover common responses of P. repens populations to drought, and climatic drivers of population differentiation in functional traits, gene expression and their plasticity.

Ancient DNA reveals the timing and persistence of organellar genetic bottlenecks over 3,000 years of sunflower domestication and improvement.

Here, we report a comprehensive paleogenomic study of archaeological and ethnographic sunflower remains that provides significant new insights into the process of domestication of this important crop. DNA from both ancient and historic contexts yielded high proportions of endogenous DNA, and although archaeological DNA was found to be highly degraded, it still provided sufficient coverage to analyze genetic changes over time. Shotgun sequencing data from specimens from the Eden's Bluff archaeological site in Arkansas yielded organellar DNA sequence from specimens up to 3,100 years old. Their sequences match those of modern cultivated sunflowers and are consistent with an early domestication bottleneck in this species. Our findings also suggest that recent breeding of sunflowers has led to a loss of genetic diversity that was present only a century ago in Native American landraces. These breeding episodes also left a profound signature on the mitochondrial and plastid haplotypes in cultivars, as two types were intentionally introduced from other Helianthus species for crop improvement. These findings gained from ancient and historic sunflower specimens underscore how future in-depth gene-based analyses can advance our understanding of the pace and targets of selection during the domestication of sunflower and other crop species.

Isolation by distance and isolation by environment contribute to population differentiation in Protea repens (Proteaceae L.), a widespread South African species.

PREMISE OF THE STUDY: The Cape Floristic Region (CFR) of South Africa is renowned for its botanical diversity, but the evolutionary origins of this diversity remain controversial. Both neutral and adaptive processes have been implicated in driving diversification, but population-level studies of plants in the CFR are rare. Here, we investigate the limits to gene flow and potential environmental drivers of selection in Protea repens L. (Proteaceae L.), a widespread CFR species. METHODS: We sampled 19 populations across the range of P. repens and used genotyping by sequencing to identify 2066 polymorphic loci in 663 individuals. We used a Bayesian FST outlier analysis to identify single-nucleotide polymorphisms (SNPs) marking genomic regions that may be under selection; we used those SNPs to identify potential drivers of selection and excluded them from analyses of gene flow and genetic structure. RESULTS: A pattern of isolation by distance suggested limited gene flow between nearby populations. The populations of P. repens fell naturally into two or three groupings, which corresponded to an east-west split. Differences in rainfall seasonality contributed to diversification in highly divergent loci, as do barriers to gene flow that have been identified in other species. CONCLUSIONS: The strong pattern of isolation by distance is in contrast to the findings in the only other widespread species in the CFR that has been similarly studied, while the effects of rainfall seasonality are consistent with well-known patterns. Assessing the generality of these results will require investigations of other CFR species.

Identification of the Submergence Tolerance QTL Come Quick Drowning1 (CQD1) in Arabidopsis thaliana.

Global climate change is predicted to increase water precipitation fluctuations and lead to localized prolonged floods in agricultural fields and natural plant communities. Thus, understanding the genetic basis of submergence tolerance is crucial in order to improve plant survival under these conditions. In this study, we performed a quantitative trait locus (QTL) analysis in Arabidopsis to identify novel candidate genes for increased submergence tolerance by using Kas-1 and Col (gl1) parental accessions and their derived recombinant inbred lines (RILs). We measured survival after submergence in dark for a 13-day period and used median lethal time, LT50 values for the QTL analysis. A major QTL, the Come Quick, Drowning (CQD1) locus, was detected in 2 independent experiments on the lower arm of chromosome 5 involved in higher submergence tolerance in the parental accession Kas-1. For fine-mapping, we then constructed near isogenic lines (NILs) by backcrossing the CQD1 QTL region. We also analyzed QTL regions related to size, leaf number, flowering, or survival in darkness and none of the QTL related to these traits overlapped with CQD1. The submergence tolerance QTL, CQD1, region detected in this study includes genes that have potential to be novel candidates effecting submergence tolerance such as trehalose-6-phosphate phosphatase and respiratory burst oxidase protein D. Gene expression and functional analysis for these genes under submergence would reveal the significance of these novel candidates and provide new perspectives for understanding genetic basis of submergence tolerance.

Variation in Arabidopsis flooding responses identifies numerous putative "tolerance genes".

Plant survival in flooded environments requires a combinatory response to multiple stress conditions such as limited light availability, reduced gas exchange and nutrient uptake. The ability to fine-tune the molecular response at the transcriptional and/or post-transcriptional level that can eventually lead to metabolic and anatomical adjustments are the underlying requirements to confer tolerance. Previously, we compared the transcriptomic adjustment of submergence tolerant, intolerant accessions and identified a core conserved and genotype-specific response to flooding stress, identifying numerous 'putative' tolerance genes. Here, we performed genome wide association analyses on 81 natural Arabidopsis accessions that identified 30 additional SNP markers associated with flooding tolerance. We argue that, given the many genes associated with flooding tolerance in Arabidopsis, improving resistance to submergence requires numerous genetic changes.

Transcriptomes of Eight Arabidopsis thaliana Accessions Reveal Core Conserved, Genotype- and Organ-Specific Responses to Flooding Stress.

Climate change has increased the frequency and severity of flooding events, with significant negative impact on agricultural productivity. These events often submerge plant aerial organs and roots, limiting growth and survival due to a severe reduction in light reactions and gas exchange necessary for photosynthesis and respiration, respectively. To distinguish molecular responses to the compound stress imposed by submergence, we investigated transcriptomic adjustments to darkness in air and under submerged conditions using eight Arabidopsis (Arabidopsis thaliana) accessions differing significantly in sensitivity to submergence. Evaluation of root and rosette transcriptomes revealed an early transcriptional and posttranscriptional response signature that was conserved primarily across genotypes, although flooding susceptibility-associated and genotype-specific responses also were uncovered. Posttranscriptional regulation encompassed darkness- and submergence-induced alternative splicing of transcripts from pathways involved in the alternative mobilization of energy reserves. The organ-specific transcriptome adjustments reflected the distinct physiological status of roots and shoots. Root-specific transcriptome changes included marked up-regulation of chloroplast-encoded photosynthesis and redox-related genes, whereas those of the rosette were related to the regulation of development and growth processes. We identified a novel set of tolerance genes, recognized mainly by quantitative differences. These included a transcriptome signature of more pronounced gluconeogenesis in tolerant accessions, a response that included stress-induced alternative splicing. This study provides organ-specific molecular resolution of genetic variation in submergence responses involving interactions between darkness and low-oxygen constraints of flooding stress and demonstrates that early transcriptome plasticity, including alternative splicing, is associated with the ability to cope with a compound environmental stress.

Transcriptome sequencing reveals population differentiation in gene expression linked to functional traits and environmental gradients in the South African shrub Protea repens.

Understanding the environmental and genetic mechanisms underlying locally adaptive trait variation across the ranges of species is a major focus of evolutionary biology. Combining transcriptome sequencing with common garden experiments on populations spanning geographical and environmental gradients holds promise for identifying such mechanisms. The South African shrub Protea repens displays diverse phenotypes in the wild along drought and temperature gradients. We grew plants from seeds collected at 19 populations spanning this species' range, and sequenced the transcriptomes of these plants to reveal gene pathways associated with adaptive trait variation. We related expression in co-expressed gene networks to trait phenotypes measured in the common garden and to source population climate. We found that expression in gene networks correlated with source-population environment and with plant traits. In particular, the activity of gene networks enriched for growth related pathways correlated strongly with source site minimum winter temperature and with leaf size, stem diameter and height in the garden. Other gene networks with enrichments for photosynthesis related genes showed associations with precipitation. Our results strongly suggest that this species displays population-level differences in gene expression that have been shaped by source population site climate, and that are reflected in trait variation along environmental gradients.

Extreme flooding tolerance in Rorippa.

Low oxygen stress imposed by floods creates a strong selection force shaping plant ecosystems in flood-prone areas. Plants inhabiting these environments adopt various adaptations and survival strategies to cope with increasing water depths. Two Rorippa species, R. sylvestris and R. amphibia that grow in naturally flooded areas, have high submergence tolerance achieved by the so-called quiescence and escape strategies, respectively. In order to dissect the molecular mechanisms involved in these strategies, we investigated submergence-induced changes in gene expression in flooded roots of Rorippa species. There was a higher induction of glycolysis and fermentation genes and faster carbohydrate reduction in R. amphibia, indicating a higher demand for energy potentially leading to faster mortality by starvation. Moreover, R. sylvestris showed induction of genes improving submergence tolerance, potentially enhancing survival in prolonged floods. Additionally, we compared transcript profiles of these 2 tolerant species to relatively intolerant Arabidopsis and found that only Rorippa species induced various inorganic pyrophosphate dependent genes, alternatives to ATP demanding pathways, thereby conserving energy, and potentially explaining the difference in flooding survival between Rorippa and Arabidopsis.

Group VII ethylene response factor diversification and regulation in four species from flood-prone environments.

Flooding events negatively affect plant performance and survival. Flooding gradients thereby determine the dynamics in vegetation composition and species abundance. In adaptation to flooding, the group VII Ethylene Response Factor genes (ERF-VIIs) play pivotal roles in rice and Arabidopsis through regulation of anaerobic gene expression and antithetical survival strategies. We investigated if ERF-VIIs have a similar role in mediating survival strategies in eudicot species from flood-prone environments. Here, we studied the evolutionary origin and regulation of ERF-VII transcript abundance and the physiological responses in species from two genera of divergent taxonomic lineages (Rumex and Rorippa). Synteny analysis revealed that angiosperm ERF-VIIs arose from two ancestral loci and that subsequent diversification and duplication led to the present ERF-VII variation. We propose that subtle variation in the regulation of ERF-VII transcript abundance could explain variation in tolerance among Rorippa species. In Rumex, the main difference in flood tolerance correlated with the genetic variation in ERF-VII genes. Large transcriptional differences were found by comparing the two genera: darkness and dark submergence-induced Rumex ERF-VIIs, whereas HRE2 expression was increased in submerged Rorippa roots. We conclude that the involvement of ERF-VIIs in flooding tolerance developed in a phylogenetic-dependent manner, with subtle variations within taxonomic clades.

Root transcript profiling of two Rorippa species reveals gene clusters associated with extreme submergence tolerance.

Complete submergence represses photosynthesis and aerobic respiration, causing rapid mortality in most terrestrial plants. However, some plants have evolved traits allowing them to survive prolonged flooding, such as species of the genus Rorippa, close relatives of Arabidopsis (Arabidopsis thaliana). We studied plant survival, changes in carbohydrate and metabolite concentrations, and transcriptome responses to submergence of two species, Rorippa sylvestris and Rorippa amphibia. We exploited the close relationship between Rorippa species and the model species Arabidopsis by using Arabidopsis GeneChip microarrays for whole-genome transcript profiling of roots of young plants exposed to a 24-h submergence treatment or air. A probe mask was used based on hybridization of genomic DNA of both species to the arrays, so that weak probe signals due to Rorippa species/Arabidopsis mismatches were removed. Furthermore, we compared Rorippa species microarray results with those obtained for roots of submerged Arabidopsis plants. Both Rorippa species could tolerate deep submergence, with R. sylvestris surviving much longer than R. amphibia. Submergence resulted in the induction of genes involved in glycolysis and fermentation and the repression of many energy-consuming pathways, similar to the low-oxygen and submergence response of Arabidopsis and rice (Oryza sativa). The qualitative responses of both Rorippa species to submergence appeared roughly similar but differed quantitatively. Notably, glycolysis and fermentation genes and a gene encoding sucrose synthase were more strongly induced in the less tolerant R. amphibia than in R. sylvestris. A comparison with Arabidopsis microarray studies on submerged roots revealed some interesting differences and potential tolerance-related genes in Rorippa species.

Wait or escape? Contrasting submergence tolerance strategies of Rorippa amphibia, Rorippa sylvestris and their hybrid.

BACKGROUND AND AIMS: Differential responses of closely related species to submergence can provide insight into the evolution and mechanisms of submergence tolerance. Several traits of two wetland species from habitats with contrasting flooding regimes, Rorippa amphibia and Rorippa sylvestris, as well as F(1) hybrid Rorippa × anceps were analysed to unravel mechanisms underlying submergence tolerance. METHODS: In the first submergence experiment (lasting 20 d) we analysed biomass, stem elongation and carbohydrate content. In the second submergence experiment (lasting 3 months) we analysed survival and the effect of re-establishment of air contact on biomass and carbohydrate content. In a separate experiment we analysed expression of two carbohydrate catabolism genes, ADH1 and SUS1, upon re-establishment of air contact following submergence. KEY RESULTS: All plants had low mortality even after 3 months of submergence. Rorippa sylvestris was characterized by 100 % survival and higher carbohydrate levels coupled with lower ADH1 gene expression as well as reduced growth compared with R. amphibia. Rorippa amphibia and the hybrid elongated their stems but this did not pay-off in higher survival when plants remained submerged. Only R. amphibia and the hybrid benefited in terms of increased biomass and carbohydrate accumulation upon re-establishing air contact. CONCLUSIONS: Results demonstrate contrasting 'escape' and 'quiescence' strategies between Rorippa species. Being a close relative of arabidopsis, Rorippa is an excellent model for future studies on the molecular mechanism(s) controlling these strategies.