Mapping of genes controlling aluminum tolerance in rice: comparison of different genetic backgrounds

Aluminum toxicity is the main factor limiting the productivity of crop plants in acid soils, particularly in the tropics and subtropics. In this study, a doubled-haploid population derived from the rice ( Oryza sativa L.) breeding lines CT9993 and IR62266 was used to map genes controlling Al tolerance. A genetic linkage map consisting of 280 DNA markers (RFLP, AFLP and SSR) was constructed to determine the position and nature of quantitative trait loci (QTLs) affecting Al tolerance. Three characters - control root length (CRL), Al-stressed root length (SRL) and root length ratio (RR) - were evaluated for the DH lines and the parents at the seedling stage in nutrient solution. A total of 20 QTLs controlling root growth under Al stress and control conditions were detected and distributed over 10 of the 12 rice chromosomes, reflecting multigenic control of these traits. The two QTLs of largest effect, qALRR-1-1 and qALRR-8 for root length ratio (a measurement of Al tolerance) were localized on chromosomes 1 and 8, respectively. Three other QTLs in addition to qALRR-8 were apparently unique in the CT9993 x IR62266 mapping population, which may explain the high level of Al tolerance in CT9993. Comparative mapping identified a conserved genomic region on chromosome 1 associated with Al tolerance across three rice genetic backgrounds. This region provides an important starting point for isolating genes responsible for different mechanisms of aluminum tolerance and understanding the genetic nature of this trait in rice and other cereals.     

QTLs and epistasis for aluminum tolerance in rice (Oryza sativa L.) at different seedling stages

To investigate the genetic background for aluminum (Al) tolerance in rice, a recombinant inbred (RI) population, derived from a cross between an Al-sensitive lowland indica rice variety IR1552 and an Al-tolerant upland japonica rice variety Azucena, was used in culture solution. A molecular linkage map, together with 104 amplified fragment length polymorphism (AFLP) markers and 103 restriction fragment length polymorphism (RFLP) markers, was constructed to map quantitative trait loci (QTLs) and epistatic loci for Al tolerance based on the segregation for relative root length (RRL) in the population. RRL was measured after stress for 2 and 4 weeks at a concentration of 1mM of Al³⁺ and a control with a pH 4.0, respectively. Two QTLs were detected at both the 2nd and the 4th weeks on chromosomes 1 and 12 from unconditional mapping, while the QTL on chromosome 1 was only detected at the 2nd stress week from conditional mapping. The effect of the QTL on chromosome 12 was increased with an increase of the stress period from 2 to 4 weeks. The QTL on chromosome 1 was expressed only at the earlier stress, but its contribution to tolerance was prolonged during growth. At least one different QTL was detected at the different stress periods. Mean comparisons between marker genotypic classes indicated that the positive alleles at the QTLs were from the Al-tolerant upland rice Azucena. An important heterozygous non-allelic interaction on Al tolerance was found. The results indicated that tolerance in the younger seedlings was predominantly controlled by an additive effect, while an epistatic effect was more important to the tolerance in older seedlings; additionally the detected QTLs may be multiple allelic loci for Al tolerance and phosphorus-uptake efficiency, or for Al and Fe²⁺ tolerance. Received: 29 July 1999 / Accepted: 13 October 1999     

Organic acid exudation as an aluminum-tolerance mechanism in maize (Zea mays L.)

In this study, the role of root organic acid synthesis and exudation in the mechanism of aluminum tolerance was examined in Al-tolerant (South American 3) and Al-sensitive (Tuxpeño and South American 5) maize genotypes. In a growth solution containing 6 M Al³⁺, Tuxpeño and South American 5 were found to be two- and threefold more sensitive to Al than South American 3. Root organic acid content and organic acid exudation from the entire root system into the bulk solution were investigated via high-performance liquid chromatographic analysis while exudates collected separately from the root apex or a mature root region (using a dividedroot-chamber technique) were analyzed with a more-sensitive ion chromatography system. In both the Al-tolerant and Al-sensitive lines, Al treatment significantly increased the total root content of organic acids, which was likely the result of Al stress and not the cause of the observed differential Al tolerance. In the absence of Al, small amounts of citrate were exuded into the solution bathing the roots. Aluminum exposure triggered a stimulation of citrate release in the Al-tolerant but not in the Al-sensitive genotypes; this response was localized to the root apex of the Al-tolerant genotype. Additionally, Al exposure triggered the release of phosphate from the root apex of the Al-tolerant genotype. The same solution Al³⁺ activity that elicited the maximum difference in Al sensitivity between Al-tolerant and Al-sensitive genotypes also triggered maximal citrate release from the root apex of the Al-tolerant line. The significance of citrate as a potential detoxifier for aluminum is discussed. It is concluded that organic acid release by the root apex could be an important aspect of Al tolerance in maize.Abbreviations SA3 South American 3, an Al-tolerant maize cultivar - SA5 South American 5, an Al-sensitive maize cultivar The authors would like to express their appreciation to Drs. John Thompson, Ross Welch and Mr. Stephen Schaefer for their training and guidance in the use of the chromatography systems. This work was supported by a Swiss National Science Foundation Fellowship to Didier Pellet, and U.S. Department of Agriculture/National Research Initiative Competitive Grant 93-37100-8874 to Leon Kochian. We would also like to thank Drs. S. Pandey and E. Ceballos from the CIMMYT Regional office at CIAT Cali, Colombia for providing seed for the maize varieties and inbred line.     

Plant improvement for tolerance to aluminum in acid soils – a review

Development of acid soils that limit crop production is an increasing problem worldwide. Many factors contribute to phytotoxicity of these soils, however, in acid soils with a high mineral content, aluminum (Al) is the major cause of toxicity. The target of Al toxicity is the root tip, in which Al exposure causes inhibition of cell elongation and cell division, leading to root stunting accompanied by reduced water and nutrient uptake. Natural variation for Al tolerance has been identified in many crop species and in some crops tolerance to Al has been introduced into productive, well-adapted varieties. Aluminum tolerance appears to be a complex multigenic trait. Selection methodology remains a limiting factor in variety development as all methods have particular drawbacks. Molecular markers have been associated with Al tolerance genes or quantitative trait loci in Arabidopsis and in several crops, which should facilitate development of additional tolerant varieties. A variety of genes have been identified that are induced or repressed upon Al exposure. Most induced genes characterized so far are not specific to Al exposure but are also induced by other stress conditions. Ectopic over-expression of some of these genes has resulted in enhanced Al tolerance. Additionally, expression of genes involved in organic acid synthesis has resulted in enhanced production of organic acids and an associated increase in Al tolerance. This review summarizes the three main approaches that have been taken to develop crops with Al tolerance: recurrent selection and breeding, development of Al tolerant somaclonal variants and ectopic expression of transgenes to reduce Al uptake or limit damage to cells by Al.     

Comparative mapping of a major aluminum tolerance gene in sorghum and other species in the poaceae

In several crop species within the Triticeae tribe of the grass family Poaceae, single major aluminum (Al) tolerance genes have been identified that effectively mitigate Al toxicity, a major abiotic constraint to crop production on acidic soils. However, the trait is quantitatively inherited in species within other tribes, and the possible ancestral relationships between major Al tolerance genes and QTL in the grasses remain unresolved. To help establish these relationships, we conducted a molecular genetic analysis of Al tolerance in sorghum and integrated our findings with those from previous studies performed in crop species belonging to different grass tribes. A single locus, AltSB, was found to control Al tolerance in two highly Al tolerant sorghum cultivars. Significant macrosynteny between sorghum and the Triticeae was observed for molecular markers closely linked to putatively orthologous Al tolerance loci present in the group 4 chromosomes of wheat, barley, and rye. However, AltSB was not located within the homeologous region of sorghum but rather mapped near the end of sorghum chromosome 3. Thus, AltSB not only is the first major Al tolerance gene mapped in a grass species that does not belong to the Triticeae, but also appears to be different from the major Al tolerance locus in the Triticeae. Intertribe map comparisons suggest that a major Al tolerance QTL on rice chromosome 1 is likely to be orthologous to AltSB, whereas another rice QTL on chromosome 3 is likely to correspond to the Triticeae group 4 Al tolerance locus. Therefore, this study demonstrates a clear evolutionary link between genes and QTL encoding the same trait in distantly related species within a single plant family.     

Physiological basis of reduced Al tolerance in ditelosomic lines of Chinese Spring wheat

Aluminum tolerance was assessed in the moderately Al-tolerant wheat (Triticum aestivum L.) cultivar Chinese Spring and a set of ditelosomic lines derived from Chinese Spring. Three ditelosomic lines lacking chromosome arms 4DL, 5AS and 7AS, respectively, exhibited decreased Al tolerance relative to the euploid parent Chinese Spring based on reduced root growth in Al-containing solutions. The physiological basis of the reduced Al tolerance was investigated. Measurements by inductively coupled argon plasma mass spectroscopy of root apical Al accumulation demonstrated that two of these three lines had a decreased ability to exclude Al from the root apex, the site of Al phytotoxicity. As Al-induced malate exudation has been suggested to be an important physiological mechanism of Al tolerance in wheat, this parameter was quantified and malate exudation was shown to be smaller in all three deletion lines compared with Chinese Spring. These results suggest that the decreased Al tolerance in at least two of the three ditelosomic lines is due to the loss of different genes independently influencing a single Al-tolerance mechanism, rather than to the loss of genes encoding alternative Al-tolerance mechanisms. Received: 3 July 2000 / Accepted: 9 August 2000     

铝胁迫下水稻幼苗根系的生理特性

以4叶1心期的水稻幼苗为材料,在水培条件下,研究了0、0.5和7.5 mmol·L^-1Al胁迫下水稻Al敏感品种IR24、耐Al品种金优725和两优培九幼苗根系的生理特性.结果表明：在7.5 mmol·L^-1Al胁迫下,耐Al品种根系活力下降幅度远小于Al敏感品种,Al敏感品种H₂O₂含量较Al耐性品种高.在Al胁迫下,各品种根系线粒体中CAT的增加幅度较小;IR24和金优725根系的线粒体POD活性随Al胁迫浓度的增加先升后降,两优培九POD活性则呈增加趋势;IR24根系线粒体APX活性随Al胁迫浓度的增加先升后降,金优725和两优培九的线粒体APX活性则呈上升趋势.随Al胁迫浓度的增加,各品种根系谷氨酸含量先增加后下降,柠檬酸含量下降,磷酸烯醇式丙酮酸含量增加.Al敏感品种抗氰呼吸速率占总呼吸的比率较耐Al品种明显降低.     

Interactive regulation of nitrogen and aluminum in rice

Despite many studies on the high aluminum (Al) tolerance of rice (Oryza sativa), its exact mechanisms remain largely unknown. It is also unclear why Al improves growth of some plants. Our research on interactions between nitrogen (N) and Al may help to understand these phenomena. Previously, we found that ammonium-supplemented rice was more Al tolerant than nitrate-supplemented rice. Furthermore, Al-tolerant rice varieties preferred ammonium, while Al-sensitive ones preferred nitrate; in fact, Al tolerance was significantly correlated with the ammonium/nitrate preference among rice varieties. Al even enhanced growth of ammonium-supplemented rice, while it inhibited growth of nitrate-supplemented rice. Based on our own and other reports on N-Al interactions, we propose that intermediate products of N metabolism may play a role in rice Al tolerance. Al-enhanced ammonium utilization may explain why Al promotes growth of some plants, since Al often coexists with higher levels of ammonium than nitrate in acid soils.     

A wheat gene encoding an aluminum-activated malate transporter

The major constraint to plant growth in acid soils is the presence of toxic aluminum (Al) cations, which inhibit root elongation. The enhanced Al tolerance exhibited by some cultivars of wheat is associated with the Al-dependent efflux of malate from root apices. Malate forms a stable complex with Al that is harmless to plants and, therefore, this efflux of malate forms the basis of a hypothesis to explain Al tolerance in wheat. Here, we report on the cloning of a wheat gene, ALMT1 (aluminum-activated malate transporter), that co-segregates with Al tolerance in F2 and F3 populations derived from crosses between near-isogenic wheat lines that differ in Al tolerance. The ALMT1 gene encodes a membrane protein, which is constitutively expressed in the root apices of the Al-tolerant line at greater levels than in the near-isogenic but Al-sensitive line. Heterologous expression of ALMT1 in Xenopus oocytes, rice and cultured tobacco cells conferred an Al-activated malate efflux. Additionally, ALMT1 increased the tolerance of tobacco cells to Al treatment. These findings demonstrate that ALMT1 encodes an Al-activated malate transporter that is capable of conferring Al tolerance to plant cells.     

Interaction between iron plaque and root border cells ameliorates aluminum toxicity of Oryza sativa differing in aluminum tolerance

Root border cells (RBCs), which are generated during plant growth and surround the root cap, and iron plaque (IP), ubiquitously formed on the root surfaces of rice, are known to alleviate aluminum (Al) toxicity. To verify the interactive effects of IP and RBCs on ameliorating Al toxicity, two rice cultivars differing in Al resistance were used to compare Al tolerance between cultivars. Additionally, root elongation, Al uptake and RBCs viability were measured as indicators of the effects of Al. The amounts of DCB-extractable Fe and Al on the root surfaces were much higher in the presence of IP than the absence. IP presence significantly decreased Al-induced inhibition of root elongation and Al contents in roots and root tips. The removal of RBCs from the root tips caused a more severe inhibition of root elongation and a higher Al accumulation in rice roots and root tips. Furthermore, root growth inhibition and Al contents in roots and root tips were significantly lower in roots with a combination of IP and RBCs than in roots with IP or RBCs only. The formation of IP on the root surface maintained higher RBCs viability and depressed mucilage exudation in an Al-tolerant rice cultivar. The results suggest that both IP and RBCs ameliorate Al toxicity, and IP has a greater capacity for Al resistance. The combination of IP and RBCs exhibited a synergistic effect associated with Al resistance.     

Genetic architecture of aluminum tolerance in rice (Oryza sativa) determined through genome-wide association analysis and QTL mapping

Aluminum (Al) toxicity is a primary limitation to crop productivity on acid soils, and rice has been demonstrated to be significantly more Al tolerant than other cereal crops. However, the mechanisms of rice Al tolerance are largely unknown, and no genes underlying natural variation have been reported. We screened 383 diverse rice accessions, conducted a genome-wide association (GWA) study, and conducted QTL mapping in two bi-parental populations using three estimates of Al tolerance based on root growth. Subpopulation structure explained 57% of the phenotypic variation, and the mean Al tolerance in Japonica was twice that of Indica. Forty-eight regions associated with Al tolerance were identified by GWA analysis, most of which were subpopulation-specific. Four of these regions co-localized with a priori candidate genes, and two highly significant regions co-localized with previously identified QTLs. Three regions corresponding to induced Al-sensitive rice mutants (ART1, STAR2, Nrat1) were identified through bi-parental QTL mapping or GWA to be involved in natural variation for Al tolerance. Haplotype analysis around the Nrat1 gene identified susceptible and tolerant haplotypes explaining 40% of the Al tolerance variation within the aus subpopulation, and sequence analysis of Nrat1 identified a trio of non-synonymous mutations predictive of Al sensitivity in our diversity panel. GWA analysis discovered more phenotype-genotype associations and provided higher resolution, but QTL mapping identified critical rare and/or subpopulation-specific alleles not detected by GWA analysis. Mapping using Indica/Japonica populations identified QTLs associated with transgressive variation where alleles from a susceptible aus or indica parent enhanced Al tolerance in a tolerant Japonica background. This work supports the hypothesis that selectively introgressing alleles across subpopulations is an efficient approach for trait enhancement in plant breeding programs and demonstrates the fundamental importance of subpopulation in interpreting and manipulating the genetics of complex traits in rice.     

Nylon Filter Arrays Reveal Differential Expression of Expressed Sequence Tags in Wheat Roots Under Aluminum Stress

To enrich differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance, cDNA subtraction libraries were generated from Al-stressed roots of two wheat (Triticum aestivum L.) nearisogenic lines (NILs) contrasting in Al-tolerance gene(s) from the Al-tolerant cultivar Atlas 66, using suppression subtractive hybridization (SSH). Expression patterns of the ESTs were investigated with nylon filter arrays containing 614 cDNA clones from the subtraction library. Gene expression profiles from macroarray analysis indicated that 25 ESTs were upregulated in the tolerant NIL in response to Al stress. The result from Northern analysis of selected upregulated ESTs was similar to that from macroarray analysis. These highly expressed ESTs showed high homology with genes involved in signal transduction, oxidative stress alleviation, membrane structure, Mg2 transportation, and other functions. Under Al stress, the Al-tolerant NIL may possess altered structure or function of the cell wall, plasma membrane, and mitochondrion. The wheat response to Al stress may involve complicated defense-related signaling and metabolic pathways.The present experiment did not detect any induced or activated genes involved in the synthesis of malate and other organic acids in wheat under Al-stress.     

大豆和水稻对铝胁迫响应的生理机制

通过水培方式,研究了铝处理对双子叶植物大豆和单子叶植物水稻根系生长、养分吸收、根系内含物及根分泌物的影响.结果表明,低铝 (10 μmol·L^-1)浸种刺激大豆种子萌发和根系的生长,对水稻无明显促进作用.铝处理增加了两种作物根系对P的吸收,降低K、Ca、Mg的吸收.水稻比大豆根系积累较少的Al和更多的P.铝胁迫条件下,大豆和水稻根系内源可溶性蛋白含量升高、可溶性酚含量下降、可溶性糖含量先上升后下降,且大豆根系内源柠檬酸含量下降明显.与大豆相比,水稻积累较低的柠檬酸和较高的可溶性蛋白、可溶性酚,但两者可溶性糖没有差异.铝处理增加大豆根系柠檬酸、可溶性蛋白、可溶性酚、可溶性糖的分泌量,且大豆分泌量显著高于水稻.在铝处理条件下,大豆根系具有较高的阳离子交换量(CEC),而水稻的CEC较低.这说明大豆和水稻对铝胁迫具有不同的生理反应,水稻的高耐铝性可能与其较高的磷吸收和较低的CEC有关,而与其根系分泌物的关系不大.     

Association and Linkage Analysis of Aluminum Tolerance Genes in Maize

Background

Aluminum (Al) toxicity is a major worldwide constraint to crop productivity on acidic soils. Al becomes soluble at low pH, inhibiting root growth and severely reducing yields. Maize is an important staple food and commodity crop in acidic soil regions, especially in South America and Africa where these soils are very common. Al exclusion and intracellular tolerance have been suggested as two important mechanisms for Al tolerance in maize, but little is known about the underlying genetics.

Methodology

An association panel of 282 diverse maize inbred lines and three F₂ linkage populations with approximately 200 individuals each were used to study genetic variation in this complex trait. Al tolerance was measured as net root growth in nutrient solution under Al stress, which exhibited a wide range of variation between lines. Comparative and physiological genomics-based approaches were used to select 21 candidate genes for evaluation by association analysis.

Conclusions

Six candidate genes had significant results from association analysis, but only four were confirmed by linkage analysis as putatively contributing to Al tolerance: Zea mays Alt_SB like (ZmASL), Zea mays aluminum-activated malate transporter2 (ALMT2), S-adenosyl-L-homocysteinase (SAHH), and Malic Enzyme (ME). These four candidate genes are high priority subjects for follow-up biochemical and physiological studies on the mechanisms of Al tolerance in maize. Immediately, elite haplotype-specific molecular markers can be developed for these four genes and used for efficient marker-assisted selection of superior alleles in Al tolerance maize breeding programs.     

Isolation and characterization of wheat aluminum-regulated genes: possible involvement of aluminum as a pathogenesis response elicitor

Using differential screening of a root tip cDNA library prepared from an Al-tolerant wheat cultivar (Triticum aestivum L. cv. Atlas-66) exposed to Al, we have isolated and characterized several wheat aluminum-regulated (War) cDNAs. Sequence comparison revealed that genes up-regulated by Al correspond to peroxidase (war4.2), cysteine proteinase (war5.2), phenylalanine-ammonia lyase (war7.2), and oxalate oxidase (war13.2). Two wheat cultivars that differ in their level of tolerance (cv. Atlas-66: tolerant, and cv. Fredrick: sensitive) were used to evaluate the relationship between the accumulation of War mRNAs and Al toxicity, as measured by root growth inhibition (RGI). The mRNA accumulation was modulated to similar levels in both cultivars compared at equivalent RGIs. This indicates that War mRNA accumulation is associated with the toxicity of Al rather than with the cultivar's tolerance. It appears that most of the genes found to be up-regulated by Al share homologies with genes induced by pathogens. This suggests that Al may act as an elicitor of a pathogenesis-related transduction pathway. The potential functions of the up-regulated war genes in cell wall strengthening and Al trapping are discussed. Received: 24 October 1997 / Accepted: 21 January 1998     

Study of aluminum toxicity by means of vital staining profiles in four cultivars of Phaseolus vulgaris L

Aluminum toxicity is a very important factor limiting crop productivity on acid soils. Early effects of aluminum toxicity comprise inhibition of cell division and effects on root elongation. The plasma membrane can be the primary target of aluminum toxicity and thus, vital staining techniques could be a powerful tool in determining effects of metal stress on the plasma membrane.

In this paper, we discuss the effects of Al on growth and membrane integrity by staining root tips with a mixture of fluorescein diacetate and propidium iodide.

The results show a good correlation between results from growth measurement and the vital staining. From the comparison of the luminosity patterns generated by vital staining it is easy to determine Al-resistant varieties, revealing this technique as a powerful and fast method for determining tolerance to Al in different varieties.     

Aluminum stress and protein synthesis in near isogenic lines of Triticum aestivum differing in aluminum tolerance

Aluminum (Al) stress was examined in three lines of wheat ( Triticum aestivum L.) by measuring root lengths, protein synthesis and protein accumulation in seedling root tips grown in a hydroponic system. An Al-sensitive, recurrent wheat parent (cv. Katepwa) showed very little root growth in low Al concentrations. In contrast, an Al-tolerant near isogenic line (Alikat) and Al-tolerant donor (cv. Maringa) had much greater root growth. Segregation data from an F₂ population (Katepwa × Alikat) showed that one major gene controlled Al tolerance based on root growth ( X ²= 0.651). All three lines showed an approximately 2-fold increase in [³⁵S]-Met incorporation in root tips after 3 days in Al and a comparable increase in root-tip dry weight. Maringa and Alikat root tips showed an increased total protein content while Katepwa root tips showed no increase in total protein content during the Al stress. Based on higher specific activities, insoluble proteins were preferentially translated in all three lines during Al stress. Proteinase activity in Katepwa root tips was 1.7-fold higher during Al stress, with Maringa and Alikat showing no change in proteinase activity. The Al-induced, increased proteinase activity in Katepwa appeared to inhibit soluble protein accumulation.