A Homolog of the Arabidopsis TIME FOR COFFEE Gene Is Involved in Nonhost Resistance to Wheat Stem Rust in Brachypodium distachyon.

Plants resist infection by pathogens using both preexisting barriers and inducible defense responses. Inducible responses are governed in a complex manner by various hormone signaling pathways. The relative contribution of hormone signaling pathways to nonhost resistance to pathogens is not well understood. In this study, we examined the molecular basis of disrupted nonhost resistance to the fungal species Puccinia graminis, which causes stem rust of wheat, in an induced mutant of the model grass Brachypodium distachyon. Through bioinformatic analysis, a 1-bp deletion in the mutant genotype was identified that introduces a premature stop codon in the gene Bradi1g24100, which is a homolog of the Arabidopsis thaliana gene TIME FOR COFFEE (TIC). In Arabidopsis, TIC is central to the regulation of the circadian clock and plays a crucial role in jasmonate signaling by attenuating levels of the transcription factor protein MYC2, and its mutational disruption results in enhanced susceptibility to the hemibiotroph Pseudomonas syringae. Our similar finding for an obligate biotroph suggests that the biochemical role of TIC in mediating disease resistance to biotrophs is conserved in grasses, and that the correct modulation of jasmonate signaling during infection by Puccinia graminis may be essential for nonhost resistance to wheat stem rust in B. distachyon.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Heritable temporal gene expression patterns correlate with metabolomic seed content in developing hexaploid oat seed.

Oat ranks sixth in world cereal production and has a higher content of health-promoting compounds compared with other cereals. However, there is neither a robust oat reference genome nor transcriptome. Using deeply sequenced full-length mRNA libraries of oat cultivar Ogle-C, a de novo high-quality and comprehensive oat seed transcriptome was assembled. With this reference transcriptome and QuantSeq 3' mRNA sequencing, gene expression was quantified during seed development from 22 diverse lines across six time points. Transcript expression showed higher correlations between adjacent time points. Based on differentially expressed genes, we identified 22 major temporal co-expression (TCoE) patterns of gene expression and revealed enriched gene ontology biological processes. Within each TCoE set, highly correlated transcripts, putatively commonly affected by genetic background, were clustered and termed genetic co-expression (GCoE) sets. Seventeen of the 22 TCoE sets had GCoE sets with median heritabilities higher than 0.50, and these heritability estimates were much higher than that estimated from permutation analysis, with no divergence observed in cluster sizes between permutation and non-permutation analyses. Linear regression between 634 metabolites from mature seeds and the PC1 score of each of the GCoE sets showed significantly lower p-values than permutation analysis. Temporal expression patterns of oat avenanthramides and lipid biosynthetic genes were concordant with previous studies of avenanthramide biosynthetic enzyme activity and lipid accumulation. This study expands our understanding of physiological processes that occur during oat seed maturation and provides plant breeders the means to change oat seed composition through targeted manipulation of key pathways.

Genomic Dissection of Nonhost Resistance to Wheat Stem Rust in Brachypodium distachyon.

The emergence of new races of Puccinia graminis f. sp. tritici, the causal pathogen of wheat stem rust, has spurred interest in developing durable resistance to this disease in wheat. Nonhost resistance holds promise to help control this and other diseases because it is durable against nonadapted pathogens. However, the genetic and molecular basis of nonhost resistance to wheat stem rust is poorly understood. In this study, the model grass Brachypodium distachyon, a nonhost of P. graminis f. sp. tritici, was used to genetically dissect nonhost resistance to wheat stem rust. A recombinant inbred line (RIL) population segregating for response to wheat stem rust was evaluated for resistance. Evaluation of genome-wide cumulative single nucleotide polymorphism allele frequency differences between contrasting pools of resistant and susceptible RILs followed by molecular marker analysis identified six quantitative trait loci (QTL) that cumulatively explained 72.5% of the variation in stem rust resistance. Two of the QTLs explained 31.7% of the variation, and their interaction explained another 4.6%. Thus, nonhost resistance to wheat stem rust in B. distachyon is genetically complex, with both major and minor QTLs acting additively and, in some cases, interacting. These findings will guide future research to identify genes essential to nonhost resistance to wheat stem rust.

Genetic Variation for Seed Metabolite Levels in Brachypodium distachyon.

Metabolite composition and concentrations in seed grains are important traits of cereals. To identify the variation in the seed metabolotypes of a model grass, namely Brachypodium distachyon, we applied a widely targeted metabolome analysis to forty inbred lines of B. distachyon and examined the accumulation patterns of 183 compounds in the seeds. By comparing the metabolotypes with the population structure of these lines, we found signature metabolites that represent different accumulation patterns for each of the three B. distachyon subpopulations. Moreover, we found that thirty-seven metabolites exhibited significant differences in their accumulation between the lines Bd21 and Bd3-1. Using a recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21, we identified the quantitative trait loci (QTLs) linked with this variation in the accumulation of thirteen metabolites. Our metabolite QTL analysis illustrated that different genetic factors may presumably regulate the accumulation of 4-pyridoxate and pyridoxamine in vitamin B6 metabolism. Moreover, we found two QTLs on chromosomes 1 and 4 that affect the accumulation of an anthocyanin, chrysanthemin. These QTLs genetically interacted to regulate the accumulation of this compound. This study demonstrates the potential for metabolite QTL mapping in B. distachyon and provides new insights into the genetic dissection of metabolomic traits in temperate grasses.

Genetic Architecture of Flowering-Time Variation in Brachypodium distachyon.

The transition to reproductive development is a crucial step in the plant life cycle, and the timing of this transition is an important factor in crop yields. Here, we report new insights into the genetic control of natural variation in flowering time in Brachypodium distachyon, a nondomesticated pooid grass closely related to cereals such as wheat (Triticum spp.) and barley (Hordeum vulgare L.). A recombinant inbred line population derived from a cross between the rapid-flowering accession Bd21 and the delayed-flowering accession Bd1-1 were grown in a variety of environmental conditions to enable exploration of the genetic architecture of flowering time. A genotyping-by-sequencing approach was used to develop SNP markers for genetic map construction, and quantitative trait loci (QTLs) that control differences in flowering time were identified. Many of the flowering-time QTLs are detected across a range of photoperiod and vernalization conditions, suggesting that the genetic control of flowering within this population is robust. The two major QTLs identified in undomesticated B. distachyon colocalize with VERNALIZATION1/PHYTOCHROME C and VERNALIZATION2, loci identified as flowering regulators in the domesticated crops wheat and barley. This suggests that variation in flowering time is controlled in part by a set of genes broadly conserved within pooid grasses.

Subgenome-specific assembly of vitamin E biosynthesis genes and expression patterns during seed development provide insight into the evolution of oat genome.

Vitamin E is essential for humans and thus must be a component of a healthy diet. Among the cereal grains, hexaploid oats (Avena sativa L.) have high vitamin E content. To date, no gene sequences in the vitamin E biosynthesis pathway have been reported for oats. Using deep sequencing and orthology-guided assembly, coding sequences of genes for each step in vitamin E synthesis in oats were reconstructed, including resolution of the sequences of homeologs. Three homeologs, presumably representing each of the three oat subgenomes, were identified for the main steps of the pathway. Partial sequences, likely representing pseudogenes, were recovered in some instances as well. Pairwise comparisons among homeologs revealed that two of the three putative subgenome-specific homeologs are almost identical for each gene. Synonymous substitution rates indicate the time of divergence of the two more similar subgenomes from the distinct one at 7.9-8.7 MYA, and a divergence between the similar subgenomes from a common ancestor 1.1 MYA. A new proposed evolutionary model for hexaploid oat formation is discussed. Homeolog-specific gene expression was quantified during oat seed development and compared with vitamin E accumulation. Homeolog expression largely appears to be similar for most of genes; however, for some genes, homoeolog-specific transcriptional bias was observed. The expression of HPPD, as well as certain homoeologs of VTE2 and VTE4, is highly correlated with seed vitamin E accumulation. Our findings expand our understanding of oat genome evolution and will assist efforts to modify vitamin E content and composition in oats.

Genome diversity in Brachypodium distachyon: deep sequencing of highly diverse inbred lines.

Brachypodium distachyon is small annual grass that has been adopted as a model for the grasses. Its small genome, high-quality reference genome, large germplasm collection, and selfing nature make it an excellent subject for studies of natural variation. We sequenced six divergent lines to identify a comprehensive set of polymorphisms and analyze their distribution and concordance with gene expression. Multiple methods and controls were utilized to identify polymorphisms and validate their quality. mRNA-Seq experiments under control and simulated drought-stress conditions, identified 300 genes with a genotype-dependent treatment response. We showed that large-scale sequence variants had extremely high concordance with altered expression of hundreds of genes, including many with genotype-dependent treatment responses. We generated a deep mRNA-Seq dataset for the most divergent line and created a de novo transcriptome assembly. This led to the discovery of >2400 previously unannotated transcripts and hundreds of genes not present in the reference genome. We built a public database for visualization and investigation of sequence variants among these widely used inbred lines.

A spontaneous segmental deletion from chromosome arm 3DL enhances Fusarium head blight resistance in wheat.

Much effort has been directed at identifying sources of resistance to Fusarium head blight (FHB) in wheat. We sought to identify molecular markers for what we hypothesized was a new major FHB resistance locus originating from the wheat cultivar 'Freedom' and introgressed into the susceptible wheat cultivar 'USU-Apogee'. An F2:3 mapping population from a cross between Apogee and A30, its BC4 near-isoline exhibiting improved FHB resistance, was evaluated for resistance. The distribution of FHB resistance in the population approximated a 1:3 moderately resistant : moderately susceptible + susceptible ratio. Separate disease evaluations established that A30 accumulated less deoxynivalenol and yielded a greater proportion of sound grain than Apogee. Molecular mapping revealed that the FHB resistance of A30 is associated with molecular markers on chromosome arm 3DL that exhibit a null phenotype in A30 but are present in both Apogee and Freedom, indicating a spontaneous deletion occurred during the development of A30. Aneuploid analysis revealed that the size of the deleted segment is approximately 19% of the arm's length. Our results suggest that the deleted interval of chromosome arm 3DL in Apogee may harbor FHB susceptibility genes that promote disease spread in infected spikes, and that their elimination increases FHB resistance in a novel manner.

Brachypodium distachyon-Cochliobolus sativus Pathosystem is a New Model for Studying Plant-Fungal Interactions in Cereal Crops.

Cochliobolus sativus (anamorph: Bipolaris sorokiniana) causes spot blotch, common root rot, and kernel blight or black point in barley and wheat. However, little is known about the molecular mechanisms underlying the pathogenicity of C. sativus or the molecular basis of resistance and susceptibility in the hosts. This study aims to establish the model grass Brachypodium distachyon as a new model for studying plant-fungus interactions in cereal crops. Six B. distachyon lines were inoculated with five C. sativus isolates. The results indicated that all six B. distachyon lines were infected by the C. sativus isolates, with their levels of resistance varying depending on the fungal isolates used. Responses ranging from hypersensitive response-mediated resistance to complete susceptibility were observed in a large collection of B. distachyon (2n=2x=10) and B. hybridum (2n=4x=30) accessions inoculated with four of the C. sativus isolates. Evaluation of an F2 population derived from the cross between two of the B. distachyon lines, Bd1-1 and Bd3-1, with isolate Cs07-47-1 showed quantitative and transgressive segregation for resistance to C. sativus, suggesting that the resistance may be governed by quantitative trait loci from both parents. The availability of whole-genome sequences of both the host (B. distachyon) and the pathogen (C. sativus) makes this pathosystem an attractive model for studying this important disease of cereal crops.

Molecular and comparative mapping of genes governing spike compactness from wild emmer wheat.

The development and morphology of the wheat spike is important because the spike is where reproduction occurs and it holds the grains until harvest. Therefore, genes that influence spike morphology are of interest from both theoretical and practical stand points. When substituted for the native chromosome 2A in the tetraploid Langdon (LDN) durum wheat background, the Triticum turgidum ssp. dicoccoides chromosome 2A from accession IsraelA confers a short, compact spike with fewer spikelets per spike compared to LDN. Molecular mapping and quantitative trait loci (QTL) analysis of these traits in a homozygous recombinant population derived from LDN × the chromosome 2A substitution line (LDNIsA-2A) indicated that the number of spikelets per spike and spike length were controlled by linked, but different, loci on the long arm of 2A. A QTL explaining most of the variation for spike compactness coincided with the QTL for spike length. Comparative mapping indicated that the QTL for number of spikelets per spike overlapped with a previously mapped QTL for Fusarium head blight susceptibility. The genes governing spike length and compactness were not orthologous to either sog or C, genes known to confer compact spikes in diploid and hexaploid wheat, respectively. Mapping and sequence analysis indicated that the gene governing spike length and compactness derived from wild emmer could be an ortholog of the barley Cly1/Zeo gene, which research indicates is an AP2-like gene pleiotropically affecting cleistogamy, flowering time, and rachis internode length. This work provides researchers with knowledge of new genetic loci and associated markers that may be useful for manipulating spike morphology in durum wheat.

Analysis and annotation of the hexaploid oat seed transcriptome.

BACKGROUND: Next generation sequencing provides new opportunities to explore transcriptomes. However, challenges remain for accurate differentiation of homoeoalleles and paralogs, particularly in polyploid organisms with no supporting genome sequence. In this study, RNA-Seq was employed to generate and characterize the first gene expression atlas for hexaploid oat. RESULTS: The software packages Trinity and Oases were used to produce a transcript assembly from nearly 134 million 100-bp paired-end reads from developing oat seeds. Based on the quality-parameters employed, Oases assemblies were superior. The Oases 67-kmer assembly, denoted dnOST (de novo Oat Seed Transcriptome), is over 55 million nucleotides in length and the average transcript length is 1,043 nucleotides. The 74.8× sequencing depth was adequate to differentiate a large proportion of putative homoeoalleles and paralogs. To assess the robustness of dnOST, we successfully identified gene transcripts associated with the biosynthetic pathways of three compounds with health-promoting properties (avenanthramides, tocols, ß-glucans), and quantified their expression. CONCLUSIONS: To our knowledge, this study provides the first direct performance comparison between two major assemblers in a polyploid organism. The workflow we developed provides a useful guide for comparable analyses in other organisms. The transcript assembly developed here is a major advance. It expands the number of oat ESTs 3-fold, and constitutes the first comprehensive transcriptome study in oat. This resource will be a useful new tool both for analysis of genes relevant to nutritional enhancement of oat, and for improvement of this crop in general.

Elevated CO2 Can Worsen Fusarium Head Blight Disease Severity in Wheat but the Fhb1 QTL Provides Reliable Disease Resistance.

Fusarium head blight (FHB) is a destructive fungal disease of wheat that causes significant economic loss due to lower yields and the contamination of grain with fungal toxins (mycotoxins), particularly deoxynivalenol (DON). FHB disease spread and mycotoxin contamination has been shown to worsen at elevated CO2, therefore, it is important to identify climate-resilient FHB resistance. This work evaluates whether wheat with the Fhb1 quantitative trait locus (QTL), the most widely deployed FHB resistance locus in wheat breeding programs, provides reliable disease resistance at elevated CO2. Near-isogenic wheat lines (NILs) derived from either a highly FHB susceptible or a more FHB resistant genetic background, with or without the Fhb1 QTL, were grown in growth chambers at ambient (400 ppm) and elevated (1000 ppm) CO2 conditions. Wheat was inoculated with Fusarium graminearum and evaluated for FHB severity. At elevated CO2, the NILs derived from more FHB-resistant wheat had increased disease spread, greater pathogen biomass and mycotoxin contamination, and lower rates of DON detoxification; this was not observed in wheat from a FHB susceptible genetic background. The Fhb1 QTL was not associated with increased disease severity in wheat grown at elevated CO2 and provided reliable disease resistance.

Grain dormancy and light quality effects on germination in the model grass Brachypodium distachyon.

• Lack of grain dormancy in cereal crops such as barley and wheat is a common problem affecting farming areas around the world, causing losses in yield and quality because of preharvest sprouting. Control of seed or grain dormancy has been investigated extensively using various approaches in different species, including Arabidopsis and cereals. However, the use of a monocot model plant such as Brachypodium distachyon presents opportunities for the discovery of new genes related to grain dormancy that are not present in modern commercial crops. • In this work we present an anatomical description of the Brachypodium caryopsis, and we describe the dormancy behaviour of six common diploid Brachypodium inbred genotypes. We also study the effect of light quality (blue, red and far-red) on germination, and analyse changes in abscisic acid levels and gene expression between a dormant and a non-dormant Brachypodium genotype. • Our results indicate that different genotypes display high natural variability in grain dormancy and that the characteristics of dormancy and germination are similar to those found in other cereals. • We propose that Brachypodium is an ideal model for studies of grain dormancy in grasses and can be used to identify new strategies for increasing grain dormancy in crop species.

QTLs for resistance to the false brome rust Puccinia brachypodii in the model grass Brachypodium distachyon L.

The potential of the model grass Brachypodium distachyon L. (Brachypodium) for studying grass-pathogen interactions is still underexploited. We aimed to identify genomic regions in Brachypodium associated with quantitative resistance to the false brome rust fungus Puccinia brachypodii . The inbred lines Bd3-1 and Bd1-1, differing in their level of resistance to P. brachypodii, were crossed to develop an F(2) population. This was evaluated for reaction to a virulent isolate of P. brachypodii at both the seedling and advanced growth stages. To validate the results obtained on the F(2), resistance was quantified in F(2)-derived F(3) families in two experiments. Disease evaluations showed quantitative and transgressive segregation for resistance. A new AFLP-based Brachypodium linkage map consisting of 203 loci and spanning 812 cM was developed and anchored to the genome sequence with SSR and SNP markers. Three false brome rust resistance QTLs were identified on chromosomes 2, 3, and 4, and they were detected across experiments. This study is the first quantitative trait analysis in Brachypodium. Resistance to P. brachypodii was governed by a few QTLs: two acting at the seedling stage and one acting at both seedling and advanced growth stages. The results obtained offer perspectives to elucidate the molecular basis of quantitative resistance to rust fungi.

Fhb1 disease resistance QTL does not exacerbate wheat grain protein loss at elevated CO2.

Fusarium head blight, a devastating cereal crop disease, can cause significant yield losses and contaminate grain with hazardous fungal toxins. Concerningly, recent evidence indicates that substantial grain protein content loss is likely to occur in wheat that is moderately resistant to head blight when it is grown at elevated CO2. Although wheat breeders in North America utilize a number of resistance sources and genes to reduce pathogen damage, the Fhb1 gene is widely deployed. To determine whether Fhb1 is associated with the protein content loss at elevated CO2, twelve near-isogenic spring wheat lines from either a susceptible or moderately susceptible genetic background, and with, or without the Fhb1 QTL, were grown at ambient and elevated CO2 conditions. The near-isogenic lines were evaluated for differences in physiology, productivity, and grain protein content. Our results showed that the Fhb1 QTL did not have any significant effect on plant growth, development, yield, or grain protein content at ambient or elevated CO2. Therefore, other factors in the moderately susceptible wheat genetic background are likely responsible for the more severe grain protein loss at elevated CO2.

Comparison of a high-density genetic linkage map to genome features in the model grass Brachypodium distachyon.

The small annual grass Brachypodium distachyon (Brachypodium) is rapidly emerging as a powerful model system to study questions unique to the grasses. Many Brachypodium resources have been developed including a whole genome sequence, highly efficient transformation and a large germplasm collection. We developed a genetic linkage map of Brachypodium using single nucleotide polymorphism (SNP) markers and an F(2) mapping population of 476 individuals. SNPs were identified by targeted resequencing of single copy genomic sequences. Using the Illumina GoldenGate Genotyping platform we placed 558 markers into five linkage groups corresponding to the five chromosomes of Brachypodium. The unusually long total genetic map length, 1,598 centiMorgans (cM), indicates that the Brachypodium mapping population has a high recombination rate. By comparing the genetic map to genome features we found that the recombination rate was positively correlated with gene density and negatively correlated with repetitive regions and sites of ancestral chromosome fusions that retained centromeric repeat sequences. A comparison of adjacent genome regions with high versus low recombination rates revealed a positive correlation between interspecific synteny and recombination rate.

An SSR-based genetic linkage map of the model grass Brachypodium distachyon.

The grass species Brachypodium distachyon (hereafter, Brachypodium) has been adopted as a model system for grasses. Here, we describe the development of a genetic linkage map of Brachypodium. The genetic linkage map was developed with an F2 population from a cross between the diploid Brachypodium lines Bd3-1 and Bd21. The map was populated with polymorphic simple sequence repeat (SSR) markers from Brachypodium expressed sequence tag (EST) and bacterial artificial chromosome (BAC) end sequences and conserved orthologous sequence (COS) markers from other grass species. The map is 1386 cM in length and consists of 139 marker loci distributed across 20 linkage groups. Five of the linkage groups exceed 100 cM in length, with the largest being 231 cM long. Assessment of colinearity between the Brachypodium linkage map and the rice genome sequence revealed significant regions of macrosynteny between the two genomes, as well as rearrangements similar to those reported in other grass comparative structural genomics studies. The Brachypodium genetic linkage map described here will serve as a new tool to pursue a range of molecular genetic analyses and other applications in this new model plant system.

Quantitative trait locus mapping of increased Fusarium head blight susceptibility associated with a wild emmer wheat chromosome.

Chromosome 2A of wild emmer wheat (Triticum turgidum var. dicoccoides) genotype Israel A increases Fusarium head blight (FHB) severity when present in durum wheat (T. turgidum var. durum) cv. Langdon (LDN). The goal of this study was to identify regions of Israel A chromosome 2A associated with this difference in resistance. A recombinant inbred chromosome line population (RICL) from a cross between LDN and the LDN-Israel A chromosome 2A substitution line [LDN(DIC-2A)] was employed for analysis. Three greenhouse FHB evaluations were completed on the RICL to obtain phenotypic data on variation for FHB resistance, and a simple sequence repeat (SSR)-based molecular map of chromosome 2A was developed. Quantitative trait locus (QTL) mapping identified a region on the long arm of chromosome 2A that was associated with FHB resistance in each independent FHB evaluation. Depending on the evaluation, the single best SSR marker in this region accounted for between 21 and 26% of the variation for FHB resistance, with the Israel A marker alleles associated with increased FHB susceptibility. The single best markers from each evaluation reside within an interval of approximately 22 cM. This study identifies one or more new QTL on chromosome 2A in tetraploid wheat that can contribute to significant variation in FHB resistance.

Lr34-mediated leaf rust resistance in wheat: transcript profiling reveals a high energetic demand supported by transient recruitment of multiple metabolic pathways.

The wheat gene Lr34 confers partial resistance to all races of Puccinia triticina, the causal agent of wheat leaf rust. However, the biological basis for the exceptional durability of Lr34 is unclear. We used the Affymetrix GeneChip Wheat Genome Array to compare transcriptional changes of near-isogenic lines of Thatcher wheat in a compatible interaction, an incompatible interaction conferred by the resistance gene Lr1, and the race-nonspecific response conditioned by Lr34 3 and 7 days postinoculation (dpi) with P. triticina. No differentially expressed genes were detected in Lr1 plants at either timepoint whereas, in the compatible Thatcher interaction, differentially expressed genes were detected only at 7 dpi. In contrast, differentially expressed genes were identified at both timepoints in P. triticina-inoculated Lr34 plants. At 3 dpi, upregulated genes associated with Lr34-mediated resistance encoded various defense and stress-related proteins, secondary metabolism enzymes, and transcriptional regulation and cellular-signaling proteins. Further, coordinated upregulation of key genes in several metabolic pathways that can contribute to increased carbon flux through the tricarboxylic cycle was detected. This indicates that Lr34-mediated resistance imposes a high energetic demand that leads to the induction of multiple metabolic responses to support cellular energy requirements. These metabolic responses were not sustained through 7 dpi, and may explain why Lr34 fails to inhibit the pathogen fully but does increase the latent period.

Physiological and molecular characterization of aluminum resistance in Medicago truncatula.

BACKGROUND: Aluminum (Al) toxicity is an important factor limiting crop production on acid soils. However, little is known about the mechanisms by which legumes respond to and resist Al stress. To explore the mechanisms of Al toxicity and resistance in legumes, we compared the impact of Al stress in Al-resistant and Al-sensitive lines of the model legume, Medicago truncatula Gaertn. RESULTS: A screen for Al resistance in 54 M. truncatula accessions identified eight Al-resistant and eight Al-sensitive lines. Comparisons of hydroponic root growth and root tip hematoxylin staining in an Al-resistant line, T32, and an Al-sensitive line, S70, provided evidence that an inducible Al exclusion mechanism occurs in T32. Transcriptional events associated with the Al resistance response were analyzed in T32 and S70 after 12 and 48 h Al treatment using oligonucleotide microarrays. Fewer genes were differentially regulated in response to Al in T32 compared to S70. Expression patterns of oxidative stress-related genes, stress-response genes and microscopic examination of Al-treated root tips suggested a lower degree of Al-induced oxidative damage to T32 root tips compared to S70. Furthermore, genes associated with cell death, senescence, and cell wall degradation were induced in both lines after 12 h of Al treatment but preferentially in S70 after 48 h of Al treatment. A multidrug and toxin efflux (MATE) transporter, previously shown to exude citrate in Arabidopsis, showed differential expression patterns in T32 and S70. CONCLUSION: Our results identified novel genes induced by Al in Al-resistant and sensitive M. truncatula lines. In T32, transcription levels of genes related to oxidative stress were consistent with reactive oxygen species production, which would be sufficient to initiate cell death of Al-accumulating cells thereby contributing to Al exclusion and root growth recovery. In contrast, transcriptional levels of oxidative stress-related genes were consistent with excessive reactive oxygen species accumulation in S70 potentially resulting in necrosis and irreversible root growth inhibition. In addition, a citrate-exuding MATE transporter could function in Al exclusion and/or internal detoxification in T32 based on Al-induced transcript localization studies. Together, our findings indicate that multiple responses likely contribute to Al resistance in M. truncatula.