Thermal stress accelerates Arabidopsis thaliana mutation rate.

Mutations are the source of both genetic diversity and mutational load. However, the effects of increasing environmental temperature on plant mutation rates and relative impact on specific mutational classes (e.g., insertion/deletion [indel] vs. single nucleotide variant [SNV]) are unknown. This topic is important because of the poorly defined effects of anthropogenic global temperature rise on biological systems. Here, we show the impact of temperature increase on Arabidopsis thaliana mutation, studying whole genome profiles of mutation accumulation (MA) lineages grown for 11 successive generations at 29°C. Whereas growth of A. thaliana at standard temperature (ST; 23°C) is associated with a mutation rate of 7 × 10-9 base substitutions per site per generation, growth at stressful high temperature (HT; 29°C) is highly mutagenic, increasing the mutation rate to 12 × 10-9 SNV frequency is approximately two- to threefold higher at HT than at ST, and HT-growth causes an ∼19- to 23-fold increase in indel frequency, resulting in a disproportionate increase in indels (vs. SNVs). Most HT-induced indels are 1-2 bp in size and particularly affect homopolymeric or dinucleotide A or T stretch regions of the genome. HT-induced indels occur disproportionately in nucleosome-free regions, suggesting that much HT-induced mutational damage occurs during cell-cycle phases when genomic DNA is packaged into nucleosomes. We conclude that stressful experimental temperature increases accelerate plant mutation rates and particularly accelerate the rate of indel mutation. Increasing environmental temperatures are thus likely to have significant mutagenic consequences for plants growing in the wild and may, in particular, add detrimentally to mutational load.

Ethylene signaling modulates Arabidopsis thaliana nitrate metabolism.

MAIN CONCLUSION: Genetic analysis reveals a previously unknown role for ethylene signaling in regulating Arabidopsis thaliana nitrogen metabolism. Nitrogen (N) is essential for plant growth, and assimilation of soil nitrate (NO3-) and ammonium ions is an important route of N acquisition. Although N import and assimilation are subject to multiple regulatory inputs, the extent to which ethylene signaling contributes to this regulation remains poorly understood. Here, our analysis of Arabidopsis thaliana ethylene signaling mutants advances that understanding. We show that the loss of CTR1 function ctr1-1 mutation confers resistance to the toxic effects of the NO3- analogue chlorate (ClO3-), and reduces the activity of the nitrate reductase (NR) enzyme of NO3- assimilation. Our further analysis indicates that the lack of the downstream EIN2 component (conferred by novel ein2 mutations) suppresses the effect of ctr1-1, restoring ClO3- sensitivity and NR activity to normal. Collectively, our observations indicate an important role for ethylene signaling in regulating Arabidopsis thaliana NO3- metabolism. We conclude that ethylene signaling enables environmentally responsive coordination of plant growth and N metabolism.

DNA mismatch repair preferentially protects genes from mutation.

Mutation is the source of genetic variation and fuels biological evolution. Many mutations first arise as DNA replication errors. These errors subsequently evade correction by cellular DNA repair, for example, by the well-known DNA mismatch repair (MMR) mechanism. Here, we determine the genome-wide effects of MMR on mutation. We first identify almost 9000 mutations accumulated over five generations in eight MMR-deficient mutation accumulation (MA) lines of the model plant species, Arabidopsis thaliana We then show that MMR deficiency greatly increases the frequency of both smaller-scale insertions and deletions (indels) and of single-nucleotide variant (SNV) mutations. Most indels involve A or T nucleotides and occur preferentially in homopolymeric (poly A or poly T) genomic stretches. In addition, we find that the likelihood of occurrence of indels in homopolymeric stretches is strongly related to stretch length, and that this relationship causes ultrahigh localized mutation rates in specific homopolymeric stretch regions. For SNVs, we show that MMR deficiency both increases their frequency and changes their molecular mutational spectrum, causing further enhancement of the GC to AT bias characteristic of organisms with normal MMR function. Our final genome-wide analyses show that MMR deficiency disproportionately increases the numbers of SNVs in genes, rather than in nongenic regions of the genome. This latter observation indicates that MMR preferentially protects genes from mutation and has important consequences for understanding the evolution of genomes during both natural selection and human tumor growth.

Environmentally responsive genome-wide accumulation of de novo Arabidopsis thaliana mutations and epimutations.

Evolution is fueled by phenotypic diversity, which is in turn due to underlying heritable genetic (and potentially epigenetic) variation. While environmental factors are well known to influence the accumulation of novel variation in microorganisms and human cancer cells, the extent to which the natural environment influences the accumulation of novel variation in plants is relatively unknown. Here we use whole-genome and whole-methylome sequencing to test if a specific environmental stress (high-salinity soil) changes the frequency and molecular profile of accumulated mutations and epimutations (changes in cytosine methylation status) in mutation accumulation (MA) lineages of Arabidopsis thaliana. We first show that stressed lineages accumulate ∼100% more mutations, and that these mutations exhibit a distinctive molecular mutational spectrum (specific increases in relative frequency of transversion and insertion/deletion [indel] mutations). We next show that stressed lineages accumulate ∼45% more differentially methylated cytosine positions (DMPs) at CG sites (CG-DMPs) than controls, and also show that while many (∼75%) of these CG-DMPs are inherited, some can be lost in subsequent generations. Finally, we show that stress-associated CG-DMPs arise more frequently in genic than in nongenic regions of the genome. We suggest that commonly encountered natural environmental stresses can accelerate the accumulation and change the profiles of novel inherited variants in plants. Our findings are significant because stress exposure is common among plants in the wild, and they suggest that environmental factors may significantly alter the rates and patterns of incidence of the inherited novel variants that fuel plant evolution.

Multiple reference genomes and transcriptomes for Arabidopsis thaliana.

Genetic differences between Arabidopsis thaliana accessions underlie the plant's extensive phenotypic variation, and until now these have been interpreted largely in the context of the annotated reference accession Col-0. Here we report the sequencing, assembly and annotation of the genomes of 18 natural A. thaliana accessions, and their transcriptomes. When assessed on the basis of the reference annotation, one-third of protein-coding genes are predicted to be disrupted in at least one accession. However, re-annotation of each genome revealed that alternative gene models often restore coding potential. Gene expression in seedlings differed for nearly half of expressed genes and was frequently associated with cis variants within 5 kilobases, as were intron retention alternative splicing events. Sequence and expression variation is most pronounced in genes that respond to the biotic environment. Our data further promote evolutionary and functional studies in A. thaliana, especially the MAGIC genetic reference population descended from these accessions.

ROS-mediated vascular homeostatic control of root-to-shoot soil Na delivery in Arabidopsis.

Sodium (Na) is ubiquitous in soils, and is transported to plant shoots via transpiration through xylem elements in the vascular tissue. However, excess Na is damaging. Accordingly, control of xylem-sap Na concentration is important for maintenance of shoot Na homeostasis, especially under Na stress conditions. Here we report that shoot Na homeostasis of Arabidopsis thaliana plants grown in saline soils is conferred by reactive oxygen species (ROS) regulation of xylem-sap Na concentrations. We show that lack of A. thaliana respiratory burst oxidase protein F (AtrbohF; an NADPH oxidase catalysing ROS production) causes hypersensitivity of shoots to soil salinity. Lack of AtrbohF-dependent salinity-induced vascular ROS accumulation leads to increased Na concentrations in root vasculature cells and in xylem sap, thus causing delivery of damaging amounts of Na to the shoot. We also show that the excess shoot Na delivery caused by lack of AtrbohF is dependent upon transpiration. We conclude that AtrbohF increases ROS levels in wild-type root vasculature in response to raised soil salinity, thereby limiting Na concentrations in xylem sap, and in turn protecting shoot cells from transpiration-dependent delivery of excess Na.

An Arabidopsis soil-salinity-tolerance mutation confers ethylene-mediated enhancement of sodium/potassium homeostasis.

High soil Na concentrations damage plants by increasing cellular Na accumulation and K loss. Excess soil Na stimulates ethylene-induced soil-salinity tolerance, the mechanism of which we here define via characterization of an Arabidopsis thaliana mutant displaying transpiration-dependent soil-salinity tolerance. This phenotype is conferred by a loss-of-function allele of ethylene overproducer1 (ETO1; mutant alleles of which cause increased production of ethylene). We show that lack of ETO1 function confers soil-salinity tolerance through improved shoot Na/K homeostasis, effected via the ethylene resistant1-constitutive triple response1 ethylene signaling pathway. Under transpiring conditions, lack of ETO1 function reduces root Na influx and both stelar and xylem sap Na concentrations, thereby restricting root-to-shoot delivery of Na. These effects are associated with increased accumulation of respiratory burst oxidase homolog F (RBOHF)-dependent reactive oxygen species in the root stele. Additionally, lack of ETO1 function leads to significant enhancement of tissue K status by an RBOHF-independent mechanism associated with elevated high-affinity K(+) TRANSPORTER5 transcript levels. We conclude that ethylene promotes soil-salinity tolerance via improved Na/K homeostasis mediated by RBOHF-dependent regulation of Na accumulation and RBOHF-independent regulation of K accumulation.

Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana.

Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)-induced single base substitutions differed substantially from those of "background" mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations.

Patterns of homoeologous gene expression shown by RNA sequencing in hexaploid bread wheat.

BACKGROUND: Bread wheat (Triticum aestivum) has a large, complex and hexaploid genome consisting of A, B and D homoeologous chromosome sets. Therefore each wheat gene potentially exists as a trio of A, B and D homoeoloci, each of which may contribute differentially to wheat phenotypes. We describe a novel approach combining wheat cytogenetic resources (chromosome substitution 'nullisomic-tetrasomic' lines) with next generation deep sequencing of gene transcripts (RNA-Seq), to directly and accurately identify homoeologue-specific single nucleotide variants and quantify the relative contribution of individual homoeoloci to gene expression. RESULTS: We discover, based on a sample comprising ~5-10% of the total wheat gene content, that at least 45% of wheat genes are expressed from all three distinct homoeoloci. Most of these genes show strikingly biased expression patterns in which expression is dominated by a single homoeolocus. The remaining ~55% of wheat genes are expressed from either one or two homoeoloci only, through a combination of extensive transcriptional silencing and homoeolocus loss. CONCLUSIONS: We conclude that wheat is tending towards functional diploidy, through a variety of mechanisms causing single homoeoloci to become the predominant source of gene transcripts. This discovery has profound consequences for wheat breeding and our understanding of wheat evolution.

Microarray-based ultra-high resolution discovery of genomic deletion mutations.

BACKGROUND: Oligonucleotide microarray-based comparative genomic hybridization (CGH) offers an attractive possible route for the rapid and cost-effective genome-wide discovery of deletion mutations. CGH typically involves comparison of the hybridization intensities of genomic DNA samples with microarray chip representations of entire genomes, and has widespread potential application in experimental research and medical diagnostics. However, the power to detect small deletions is low. RESULTS: Here we use a graduated series of Arabidopsis thaliana genomic deletion mutations (of sizes ranging from 4 bp to ~5 kb) to optimize CGH-based genomic deletion detection. We show that the power to detect smaller deletions (4, 28 and 104 bp) depends upon oligonucleotide density (essentially the number of genome-representative oligonucleotides on the microarray chip), and determine the oligonucleotide spacings necessary to guarantee detection of deletions of specified size. CONCLUSIONS: Our findings will enhance a wide range of research and clinical applications, and in particular will aid in the discovery of genomic deletions in the absence of a priori knowledge of their existence.

HANDS: a tool for genome-wide discovery of subgenome-specific base-identity in polyploids.

BACKGROUND: The analysis of polyploid genomes is problematic because homeologous subgenome sequences are closely related. This relatedness makes it difficult to assign individual sequences to the specific subgenome from which they are derived, and hinders the development of polyploid whole genome assemblies. RESULTS: We here present a next-generation sequencing (NGS)-based approach for assignment of subgenome-specific base-identity at sites containing homeolog-specific polymorphisms (HSPs): 'HSP base Assignment using NGS data through Diploid Similarity' (HANDS). We show that HANDS correctly predicts subgenome-specific base-identity at >90% of assayed HSPs in the hexaploid bread wheat (Triticum aestivum) transcriptome, thus providing a substantial increase in accuracy versus previous methods for homeolog-specific base assignment. CONCLUSION: We conclude that HANDS enables rapid and accurate genome-wide discovery of homeolog-specific base-identity, a capability having multiple applications in polyploid genomics.

Evolution of a plant growth-regulatory protein interaction specificity.

Specific protein-protein interactions (PPIs) enable biological regulation. However, the evolution of PPI specificity is little understood. Here we trace the evolution of the land-plant growth-regulatory DELLA-SLY1/GID2 PPI, revealing progressive increase in specificity of affinity of SLY1/GID2 for a particular DELLA form. While early-diverging SLY1s display relatively broad-range DELLA affinity, later-diverging SLY1s tend towards increasingly stringent affinity for a specific DELLA A' form generated by the growth-promoting phytohormone gibberellin (GA). Our novel mutational strategy reveals amino acid substitutions contributing to the evolution of Arabidopsis thaliana SLY1 A' specificity, also showing that routes permitting reversion to broader affinity became increasingly constrained over evolutionary time. We suggest that progressive affinity narrowing may be an important evolutionary driver of PPI specificity and that increase in SLY1/GID2-DELLA specificity enabled the enhanced flexibility of plant physiological environmental adaptation conferred by the GA-DELLA growth-regulatory mechanism.

The potential of stable carbon and nitrogen isotope analysis of foxtail and broomcorn millets for investigating ancient farming systems.

Foxtail and broomcorn millets are the most important crops in northern China since the early Neolithic. However, little evidence is available on how people managed these two crops in the past, especially in prehistory. Previous research on major C3 crops in western Eurasia demonstrated the potential of stable carbon and nitrogen isotope analysis of charred archaeobotanical remains to reveal the management of water and manure, respectively. Here, we evaluate the feasibility of a similar approach to C4 millets. Foxtail and broomcorn millet plants grown in pots in a greenhouse under different manuring and watering regimes were analysed to test the effects of management on stable carbon and nitrogen isotope values of grains. Stable nitrogen isotope values of both millets increased as manuring level increased, ranging from 1.7 ‰ to 5.8 ‰ in different conditions; hence, it appears a feasible tool to identify manuring practices, in agreement with results from recent field studies. However, the two millets exhibit opposing trends in stable carbon isotope values as watering level increased. The shift in stable carbon isotope values of millets is also smaller than that observed in wheat grown in the same experimental environment, making it difficult to identify millet water status archaeologically. In addition, we charred millet grains at different temperatures and for varying durations to replicate macro-botanical remains recovered archaeologically, and to evaluate the offsets in carbon and nitrogen isotope values induced by charring. We found that the stable nitrogen isotope values of foxtail millet and broomcorn millet can shift up to 1-2 ‰ when charred, while the stable carbon isotope values change less than 0.3 ‰. Overall, we demonstrate that stable nitrogen isotope values of charred foxtail and broomcorn millet seeds could provide insight into past field management practices, and both carbon and nitrogen isotope values can together inform palaeodietary reconstruction.

The gateway pDEST17 expression vector encodes a -1 ribosomal frameshifting sequence.

The attB1 site in the Gateway (Invitrogen) bacterial expression vector pDEST17, necessary for in vitro site-specific recombination, contains the sequence AAA-AAA. The sequence A-AAA-AAG within the Escherichia coli dnaX gene is recognized as 'slippery' and promotes -1 translational frameshifting. We show here, by expressing in E. coli several plant cDNAs with and without single nucleotide deletions close to the translation initiation codons, that pDEST17 is intrinsically susceptible to -1 ribosomal frameshifting at the sequence C-AAA-AAA. The deletion mutants produce correct-sized, active enzymes with a good correlation between enzyme amount and activity. We demonstrate unambiguously the frameshift through a combination of Edman degradation, MALDI-ToF mass fingerprint analysis of tryptic peptides and MALDI-ToF reflectron in-source decay (rISD) sequencing. The degree of frameshifting depends on the nature of the sequence being expressed and ranged from 25 to 60%. These findings suggest that caution should be exercised when employing pDEST17 for high-level protein expression and that the attB1 site has some potential as a tool for studying -1 frameshifting.

Characterization of Medicago truncatula (barrel medic) hydroperoxide lyase (CYP74C3), a water-soluble detergent-free cytochrome P450 monomer whose biological activity is defined by monomer-micelle association.

We describe the detailed biochemical characterization of CYP74C3 (cytochrome P450 subfamily 74C3), a recombinant plant cytochrome P450 enzyme with HPL (hydroperoxide lyase) activity from Medicago truncatula (barrel medic). Steady-state kinetic parameters, substrate and product specificities, RZ (Reinheitszahl or purity index), molar absorption coefficient, haem content, and new ligands for an HPL are reported. We show on the basis of gel filtration, sedimentation velocity (sedimentation coefficient distribution) and sedimentation equilibrium (molecular mass) analyses that CYP74C3 has low enzyme activity as a detergent-free, water-soluble, monomer. The enzyme activity can be completely restored by re-activation with detergent micelles, but not detergent monomers. Corresponding changes in the spin state equilibrium, and probably co-ordination of the haem iron, are novel for cytochrome P450 enzymes and suggest that detergent micelles have a subtle effect on protein conformation, rather than substrate presentation, which is sufficient to improve substrate binding and catalytic-centre activity by an order of magnitude. The kcat/K(m) of up to 1.6x10(8) M(-1) x s(-1) is among the highest recorded, which is remarkable for an enzyme whose reaction mechanism involves the scission of a C-C bond. We carried out both kinetic and biophysical studies to demonstrate that this effect is a result of the formation of a complex between a protein monomer and a single detergent micelle. Association with a detergent micelle rather than oligomeric state represents a new mechanism of activation for membrane-associated cytochrome P450 enzymes. Highly concentrated and monodispersed samples of detergent-free CYP74C3 protein may be well suited for the purposes of crystallization and structural resolution of the first plant cytochrome P450 enzyme.

Allene oxide synthase from Arabidopsis thaliana (CYP74A1) exhibits dual specificity that is regulated by monomer-micelle association.

We investigate the effects of detergent on the kinetics and oligomeric state of allene oxide synthase (AOS) from Arabidopsis thaliana (CYP74A1). We show that detergent-free CYP74A1 is monomeric and highly water soluble with dual specificity, but has relatively low activity. Detergent micelles promote a 48-fold increase in k(cat)/K(m) (to 5.9 x 10(7)M(-1)s(-1)) with concomitant changes in the spin state equilibrium of the haem-iron due to the binding of a single detergent micelle to the protein monomer, which is atypical of P450 enzymes. This mechanism is shown to be an important determinant of the substrate specificity of CYP74A1. CYP74A1 may be suited for structural resolution of the first plant cytochrome P450 and its 9-AOS activity and behaviour in vitro has implications for its role in planta.

HANDS2: accurate assignment of homoeallelic base-identity in allopolyploids despite missing data.

Characterization of homoeallelic base-identity in allopolyploids is difficult since homeologous subgenomes are closely related and becomes further challenging if diploid-progenitor data is missing. We present HANDS2, a next-generation sequencing-based tool that enables highly accurate (>90%) genome-wide discovery of homeolog-specific base-identity in allopolyploids even in the absence of a diploid-progenitor. We applied HANDS2 to the transcriptomes of various cruciferous plants belonging to genus Brassica. Our results suggest that the three C genomes in Brassica are more similar to each other than the three A genomes, and provide important insights into the relationships between various Brassica tetraploids and their diploid-progenitors at a single-base resolution.

Overexpressing the Multiple-Stress Responsive Gene At1g74450 Reduces Plant Height and Male Fertility in Arabidopsis thaliana.

A subset of genes in Arabidopsis thaliana is known to be up-regulated in response to a wide range of different environmental stress factors. However, not all of these genes are characterized as yet with respect to their functions. In this study, we used transgenic knockout, overexpression and reporter gene approaches to try to elucidate the biological roles of five unknown multiple-stress responsive genes in Arabidopsis. The selected genes have the following locus identifiers: At1g18740, At1g74450, At4g27652, At4g29780 and At5g12010. Firstly, T-DNA insertion knockout lines were identified for each locus and screened for altered phenotypes. None of the lines were found to be visually different from wildtype Col-0. Secondly, 35S-driven overexpression lines were generated for each open reading frame. Analysis of these transgenic lines showed altered phenotypes for lines overexpressing the At1g74450 ORF. Plants overexpressing the multiple-stress responsive gene At1g74450 are stunted in height and have reduced male fertility. Alexander staining of anthers from flowers at developmental stage 12-13 showed either an absence or a reduction in viable pollen compared to wildtype Col-0 and At1g74450 knockout lines. Interestingly, the effects of stress on crop productivity are most severe at developmental stages such as male gametophyte development. However, the molecular factors and regulatory networks underlying environmental stress-induced male gametophytic alterations are still largely unknown. Our results indicate that the At1g74450 gene provides a potential link between multiple environmental stresses, plant height and pollen development. In addition, ruthenium red staining analysis showed that At1g74450 may affect the composition of the inner seed coat mucilage layer. Finally, C-terminal GFP fusion proteins for At1g74450 were shown to localise to the cytosol.

Microarray-based optimization to detect genomic deletion mutations.

We performed array comparative genome hybridization (aCGH) analyses of five Arabidopsis thaliana mutants with genomic deletions ranging in size from 4 bp to > 5 kb. We used the Roche NimbleGen Arabidopsis CGH 3 × 720 K whole genome custom tiling array to optimize deletion detection. Details of the microarray design and hybridization data have been deposited at the NCBI GEO repository with accession number GSE55327.