Engineering greater aluminium resistance in wheat by over-expressing TaALMT1

Allele diversities of four markers specific to intron three, exon four and promoter regions of the aluminum (Al) resistance gene of wheat (Triticum aestivum L.) TaALMT1 were compared in 179 common wheat cultivars used in international wheat breeding programs. In wheat cultivars released during the last 93 years, six different promoter types were identified on the basis of allele size. A previous study showed that Al resistance was not associated with a particular coding allele for TaALMT1 but was correlated with blocks of repeated sequence upstream of the coding sequence. We verified the linkage between these promoter alleles and Al resistance in three doubled haploid and one intercross populations segregating for Al resistance. Molecular and pedigree analysis suggest that Al resistance in modern wheat germplasm is derived from several independent sources. Analysis of a population of 278 landraces and subspecies of wheat showed that most of the promoter alleles associated with Al resistance pre-existed in Europe, the Middle East and Asia prior to dispersal of cultivated germplasm around the world. Furthermore, several new promoter alleles were identified among the landraces surveyed. The TaALMT1 promoter alleles found within the spelt wheats were consistent with the hypothesis that these spelts arose on several independent occasions from hybridisations between non-free-threshing tetraploid wheats and Al-resistant hexaploid bread wheats. The strong correlation between Al resistance and Al-stimulated malate efflux from the root apices of 49 diverse wheat genotypes examined was consistent with the previous finding that Al resistance in wheat is conditioned primarily by malate efflux. These results demonstrate that the markers based on intron, exon and promoter regions of TaALMT1 can trace the inheritance of the Al resistance locus within wheat pedigrees and track Al resistance in breeding programmes.

Aluminum (Al) resistance in wheat relies on the Al-activated malate efflux from root apices, which appears to be controlled by an Al-activated anion transporter encoded by the ALMT1 gene on chromosome 4DL. Genomic regions upstream and downstream of ALMT1 in 69 wheat lines were characterized to identify patterns that might influence ALMT1 expression. The first 1,000 bp downstream of ALMT1 was conserved among the lines examined apart from the presence of a transposon-like sequence which did not correlate with Al resistance. In contrast, the first 1,000 bp upstream of the ALMT1 coding region was more variable and six different patterns could be discerned (types I-VI). Type I had the simplest structure, while the others had blocks of sequence that were duplicated or triplicated in different arrangements. A pattern emerged among the lines of non-Japanese origin such that the number of repeats in this upstream region was positively correlated with the levels of ALMT1 expression and Al resistance. In contrast, many of the Japanese lines exhibited a large variation in ALMT1 expression and Al resistance despite possessing the same type of upstream region. Although ALMT1 expression was also poorly correlated with Al-activated malate efflux in the Japanese lines, a strong correlation between malate efflux and Al resistance suggested that malate efflux was still the primary mechanism for Al resistance, and that additional genes are involved in the post-transcriptional regulation of ALMT1 function.

Erbium (Er), an element of the lanthanide series, exists as a trivalent cation in solution at pH 4.3. When root tips of a pair isogenic wheat (Triticum aestivum L.) lines were exposed to 100 μM Er containing 0.2 mM CaCl2 (pH 4.3) for 4 h, a greater amount of malate was exuded from the Al-resistant genotype (ET8) than from the Al-sensitive genotype (ES8). Previous studies using these genotypes (Delhaize et al. 1993b, Plant Physiol. 103, 695–693; Ryan et al. 1995, Planta 196, 103–110) showed that Al-activated malate efflux from root apices is a likely mechanism of Al resistance in wheat (the “malate hypothesis”). In contrast to Al, Er inhibited root growth of both genotypes equally. Er-activated malate efflux cosegregated with Al resistance in wheat seedlings derived from a cross between Al-sensitive and Al-resistant near-isogenic lines. This co-segregation suggests that the Er-stimulated malate efflux is encoded by the Al resistance locus, Alt1, and provides additional genetic evidence that the ability to exude large amounts of malate is associated with Al resistance in wheat. This is the first study to identify a cation, other than Al3+, which can activate malate release from wheat roots.

Crop production on acidic soils: overcoming aluminium toxicity and phosphorus deficiency

Aluminum (Al) toxicity is a major constraint for wheat production in acidic soils. An Al resistance gene on chromosome 4DL that traces to Brazilian wheat has been extensively studied, and can provide partial protection from Al damage. To identify potentially new sources of Al resistance, 590 wheat accessions, including elite wheat breeding lines from the United States and other American and European countries, landraces and commercial cultivars from East Asia, and synthetic wheat lines from CIMMYT, Mexico, were screened for Al resistance by measuring relative root elongation in culture with a nutrient solution containing Al, and by staining Al-stressed root tips with hematoxylin. Eighty-eight wheat accessions demonstrated at least moderate resistance to Al toxicity. Those selected lines were subjected to analysis of microsatellite markers linked to an Al resistance gene on 4DL and a gene marker for the Al-activated malate transporter (ALMT1) locus. Many of the selected Al-resistant accessions from East Asia did not have the Al-resistant marker alleles of ALMT1, although they showed Al resistance similar to the US Al-resistant cultivar, Atlas 66. Most of the cultivars derived from Jagger and Atlas 66 have the Al-resistant marker alleles of ALMT1. Cluster analysis separated the selected Al-resistant germplasm into two major clusters, labeled as Asian and American–European clusters. Potentially new germplasm of Al resistance different from those derived from Brazil were identified. Further investigation of Al resistance in those new germplasms may reveal alternative Al-resistance mechanisms in wheat.

The mechanisms of aluminum (Al) resistance in wheat and rye involve the release of citrate and malate anions from the root apices. Many of the genes controlling these processes have been identified and their responses to Al treatment described in detail. This study investigated how the major Al resistance traits of wheat and rye are transferred to triticale (x Tritosecale Wittmack) which is a hybrid between wheat and rye. We generated octoploid and hexaploid triticale lines and compared them with the parental lines for their relative resistance to Al, organic anion efflux and expression of some of the genes encoding the transporters involved. We report that the strong Al resistance of rye was incompletely transferred to octoploid and hexaploid triticale. The wheat and rye parents contributed to the Al-resistance of octoploid triticale but the phenotypes were not additive. The Al resistance genes of hexaploid wheat, TaALMT1, and TaMATE1B, were more successfully expressed in octoploid triticale than the Al resistance genes in rye tested, ScALMT1 and ScFRDL2. This study demonstrates that an important stress-tolerance trait derived from hexaploid wheat was expressed in octoploid triticale. Since most commercial triticale lines are largely hexaploid types it would be beneficial to develop techniques to generate genetically-stable octoploid triticale material. This would enable other useful traits that are present in hexaploid but not tetraploid wheat, to be transferred to triticale.

SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) is a core transcription factor that regulates the expression of aluminum (Al) resistance genes to manage Al toxicity in plants. However, the genome-wide roles of SlSTOP1 in the Al stress response of tomato (Solanum lycopersicum) remain largely unknown. Here, we report that SlSTOP1 is crucial for Al tolerance in tomato, as loss-of-function mutants of SlSTOP1 displayed hypersensitivity to Al stress. Aluminum stress had no effect on SlSTOP1 mRNA expression, but promoted accumulation of SlSTOP1 protein in the nucleus. Through integrated DNA affinity purification sequencing and RNA sequencing analysis, we identified 39 SlSTOP1-targeted Al-responsive genes, some of which are homologous to known Al resistance genes in other plant species, suggesting that these SlSTOP1-targeted genes play essential roles in Al resistance in tomato. Furthermore, using peak enrichment analysis of SlSTOP1-targeted sequences, we identified a cis-acting element bound by SlSTOP1 and validated this finding via dual-luciferase reporter and electrophoretic mobility shift assay (EMSA). Additionally, we demonstrated SlHAK5 is one of direct targets of SlSTOP1 and functionally characterized it in terms of Al stress tolerance. Compared with wild-type plants, Slhak5 mutants developed by CRISPR/Cas9 technology presented increased sensitivity to Al stress, which was associated with reduced citrate secretion from the roots. Together, our findings demonstrate that SlSTOP1 directly interacts with cis-acting elements located in the promoters of target genes involved in diverse pathways contributing to Al resistance in tomato.

Although it is well known that aluminum (Al) resistance in wheat (Triticum aestivum) is multigenic, physiological evidence for multiple mechanisms of Al resistance has not yet been documented. The role of root apical phosphate and malate exudation in Al resistance was investigated in two wheat cultivars (Al-resistant Atlas and Al-sensitive Scout) and two near-isogenic lines (Al-resistant ET3 and Al-sensitive ES3). In Atlas Al resistance is multigenic, whereas in ET3 resistance is conditioned by the single Alt1 locus. Based on root- growth experiments, Atlas was found to be 3-fold more resistant in 20 [mu]M Al than ET3. Root-exudation experiments were conducted under sterile conditions; a large malate efflux localized to the root apex was observed only in Atlas and in ET3 and only in the presence of Al (5 and 20 [mu]M). Furthermore, the more Al-resistant Atlas exhibited a constitutive phosphate release localized to the root apex. As predicted from the formation constants for the Al-malate and Al-phosphate complexes, the addition of either ligand to the root bathing solution alleviated Al inhibition of root growth in Al-sensitive Scout. These results provide physiological evidence that Al resistance in Atlas is conditioned by at least two genes. In addition to the alt locus that controls Al-induced malate release from the root apex, other genetic loci appear to control constitutive phosphate release from the apex. We suggest that both exudation processes act in concert to enhance Al exclusion and Al resistance in Atlas.

Aluminum (Al) toxicity on acidic soils significantly damages plant roots and inhibits root growth. Hence, crops intoxicated by Al become more sensitive to drought stress and mineral nutrient deficiencies, particularly phosphorus (P) deficiency, which is highly unavailable on tropical soils. Advances in our understanding of the physiological and genetic mechanisms that govern plant Al resistance have led to the identification of Al resistance genes, both in model systems and in crop species. It has long been known that Al resistance has a beneficial effect on crop adaptation to acidic soils. This positive effect happens because the root systems of Al resistant plants show better development in the presence of soil ionic Al3+ and are, consequently, more efficient in absorbing sub-soil water and mineral nutrients. This effect of Al resistance on crop production, by itself, warrants intensified efforts to develop and implement, on a breeding scale, modern selection strategies to profit from the knowledge of the molecular determinants of plant Al resistance. Recent studies now suggest that Al resistance can exert pleiotropic effects on P acquisition, potentially expanding the role of Al resistance on crop adaptation to acidic soils. This appears to occur via both organic acid (OA)- and non-OA transporters governing a joint, iron-dependent interplay between Al resistance and enhanced P uptake, via changes in root system architecture. Current research suggests this interplay to be part of a P stress response, suggesting that this mechanism could have evolved in crop species to improve adaptation to acidic soils. Should this pleiotropism prove functional in crop species grown on acidic soils, molecular breeding based on Al resistance genes may have a much broader impact on crop performance than previously anticipated. To explore this possibility, here we review the components of this putative effect of Al resistance genes on P stress responses and P nutrition to provide the foundation necessary to discuss the recent evidence suggesting pleiotropy as a genetic linkage between Al resistance and P efficiency. We conclude by exploring what may be needed to enhance the utilization of Al resistance genes to improve crop production on acidic soils.

We summarize progress made towards the development of tools and knowledge that aid the genetic improvement of aluminum (Al) resistance in two of CIAT’s (International Center for Tropical Agriculture) commodity crops; common bean ( Phaseolus vulgaris L.) and brachiariagrasses ( Brachiaria spp . Grisib ). Approximately 40% of common bean production is on acid soils where Al toxicity limits root development. Improving Al resistance, therefore, is an important breeding objective. We compared a group of 53 common bean genotypes with differing levels of adaptation to acid soils to identify Al resistant genotypes using a hydroponic screening method. Four different root traits (percent inhibition of root elongation, percent increase of average root diameter, total root length per plant and total number of root tips per plant) were identified as useful indicators for Al resistance. Application of this method to 30 accessions of scarlet runner bean (P. coccineus L.) identified three Al-resistant genotypes that could be used for introgressing Al resistance into cultivated germplasm . Work is in progress to characterize the physiological mechanisms underpinning Al resistance in common bean. This will facilitate developing simplified screening procedures and identifying quantitative trait loci ( QTLs ) contributing to Al resistance. Compared to common bean, brachiariagrasses tend to be better adapted to acid soils. Yet edaphic adaptation is an important component of CIAT’s and EMBRAPA’s ( Empresa Brasiliera de Pesquisa Agropecuaria ) breeding programs because one of the three parental genotypes is poorly adapted. We developed and incorporated into our breeding program a solution-culture technique that uses rooted stem cuttings to screen for Al resistance and root vigor, both of which are segregating in breeding populations. Physiological research into the remarkably high level of Al resistance of signalgrass (B. decumbens Stapf ) has ruled out a significant contribution of organic-acid secretion at root apices (a widespread Al-exclusion mechanism in plants). Instead, Al resistance appears to be a facet of a more generic resistance mechanism that prevents intoxication by inorganic cations , possibly as a result of the change in composition of the lipid bilayer of root cell plasma membranes. Once taken up into roots, Al appears to be complexed by low-molecular-weight ligands such as citrate. Other adaptive traits may counteract secondary effects of Al toxicity on phosphorus (P) nutrition through stimulation of P uptake or use efficiency.

This chapter aims at describing the main physiological mechanisms associated with aluminium (Al) resistance in wheat and how the research about these mechanisms has evolved to its current status. Practical aspects of phenotyping and using the molecular basis to increase Al resistance, which can be easily introduced in breeding programmes, are detailed. This chapter discusses the reliability of methods to screen root growth under Al stress, the allelic variation of genes associated with the main Al resistance mechanism in wheat, the quantitative trait loci and genomic regions that might contain minor Al tolerance genes, the use of wheat wild relatives, the uncertainties of developing transgenic wheat for greater Al resistance and the development of Al-resistant lines of durum wheat (Triticum turgidum subsp. durum).

Genotypic differences in aluminum (Al) resistance in rye (Secale cereale L.), triticale (X Triticosecale Wittmack), wheat (Triticum aestivum L.), and buckwheat (Fygopyrum esculentum Moench) were examined using a compartmental hydroponic system. Four-day-old seedlings were grown for 24 h in 0.5 mM CaCl2 (pH 4.5) containing 0 or 50 μM Al. Relative root elongation (RRE) at 50 μM Al. (as a percentage of that at 0 Al) was used as the index of Al resistance. On average, rye exhibited the highest Al resistance, followed by buckwheat, triticale, and wheat. However, triticale displayed the greatest genotypic differences. Al content in the root tips of triticale breeding lines negatively correlated with RRE (r = 0.5, P < 0.01), implying that the Al exclusion mechanism contributed to Al resistance. Furthermore, high Al resistance in buckwheat correlated well with the growth habitats of buckwheat, indicating that adaptation mechanisms giving good Al resistance have evolved in buckwheat. All of these results suggested that it is possible to obtain greater Al resistance in plants by screening current existing cultivars. The selection of new cultivars with increased Al resistance and sensitivity will provide important material for further studies exploring Al phytotoxic and resistant mechanisms.

The first confirmed mechanism for aluminum (Al) resistance in plants is encoded by the wheat (Triticum aestivum) gene, TaALMT1, on chromosome 4DL. TaALMT1 controls the Al-activated efflux of malate from roots, and this mechanism is widespread among Al-resistant genotypes of diverse genetic origins. This study describes a second mechanism for Al resistance in wheat that relies on citrate efflux. Citrate efflux occurred constitutively from the roots of Brazilian cultivars Carazinho, Maringa, Toropi, and Trintecinco. Examination of two populations segregating for this trait showed that citrate efflux was controlled by a single locus. Whole-genome linkage mapping using an F(2) population derived from a cross between Carazinho (citrate efflux) and the cultivar EGA-Burke (no citrate efflux) identified a major locus on chromosome 4BL, Xce(c), which accounts for more than 50% of the phenotypic variation in citrate efflux. Mendelizing the quantitative variation in citrate efflux into qualitative data, the Xce(c) locus was mapped within 6.3 cM of the microsatellite marker Xgwm495 locus. This linkage was validated in a second population of F(2:3) families derived from a cross between Carazinho and the cultivar Egret (no citrate efflux). We show that expression of an expressed sequence tag, belonging to the multidrug and toxin efflux (MATE) gene family, correlates with the citrate efflux phenotype. This study provides genetic and physiological evidence that citrate efflux is a second mechanism for Al resistance in wheat.

BackgroundAluminum (Al) toxicity represents a major constraint for crop production on acid soils. Rice is a high Al-resistant plant species among small-grain cereals, but its molecular mechanisms of Al resistance are not fully understood. We adopted a forward genetic screen strategy to uncover the Al-resistance mechanisms in rice. In this study, we screened an ethylmethylsulfone (EMS)-mutagenized library to isolate and characterize mutants with altered sensitivity to Al in rice.ResultsTreatment of an Al-intolerant indica variety Kasalath with 20 μM Al induced root swelling. This phenotype could be suppressed by the addition of aminoethoxyvinylglycine (AVG, an ethylene synthesis inhibitor), suggesting that increased production of ethylene is responsible for the root swelling under Al stress. By utilizing the root swelling as an indicator, we developed a highly effective method to screen Al-sensitive or -resistant mutants in rice. Through screening of ~5000 M2 lines, we identified 10 Al-sensitive mutants and one Al-resistant mutant ral1 (resistance to aluminum 1). ral1 mutant showed short root phenotype under normal growth condition, which was attributed to reduced cell elongation in the mutant. A dose-response experiment revealed that ral1 mutant was more resistant to Al than wild-type (WT) at all Al concentrations tested. The mutant was also more resistant to Al when grown in an acid soil. The mutant accumulated much lower Al in the root tips (0–1 cm) than WT. The mutant contained less Al in the cell wall of root tips than WT, whereas Al concentration in the cell sap was similar between WT and the mutant. In addition to Al, the mutant was also more resistant to Cd than WT. Quantitative RT-PCR analysis showed that the expression levels of known Al-resistance genes were not increased in the mutant compared to WT. Genetic analysis indicated that the Al-resistance phenotype in ral1 mutant was controlled by a single recessive gene mapped on the long arm of chromosome 6.ConclusionsWe have developed a highly efficient method for the screening of rice mutants with altered Al sensitivity. We identified a novel mutant ral1 resistant to Al by this screening. The increased resistance of ral1 to Al toxicity is caused by the reduced Al binding to the cell wall of root tips and the responsible gene is mapped on the long arm of chromosome 6.

Genetically determined differences in aluminum (Al) resistance exist among plant species and genotypes, and efforts to select and breed maize germplasm with higher resistance to Al have been made worldwide. This work aimed to study genotypic variation for Al resistance in maize genotypes by using three different screening techniques, to compare the results of the screening techniques, and to select genotypes with differential sensitivity to Al for further research on the mechanisms of Al resistance in maize. The effects of Al on various plant characteristics were studied with 10 maize genotypes in a series of experiments that comprised short-term (4 and 14 d) exposures to Al in culture solution (up to 100 µM Al), as well as longer-term (40 d) growth in an acid soil (soil solution pH range 3.4–4.1). Al resistance varied widely among the maize genotypes, as revealed by the different screening methods used. A screening method based on root elongation rate of seedlings growing in culture solution was effective in discriminating resistance to Al. A concentration of 40 µM Al gave the best differential responses among the 10 genotypes studied, causing reductions in root elongation rate of 10 to 68%. The best indicators of differential Al resistance were root characteristics, especially root length. Internal root concentrations of citrate and malate, however, did not reflect plant resistance to Al. The Al resistance rankings established with the screening techniques were consistent and indicated genotypes with contrasting sensitivity to Al to be used in further studies of mechanisms of Al resistance in maize.