Lyza G. Maron

Two functionally distinct members of the MATE (multi‐drug and toxic compound extrusion) family of transporters potentially underlie two major aluminum tolerance QTLs in maize

Crop yields are significantly reduced by aluminum (Al) toxicity on acidic soils, which comprise up to 50% of the worlds arable land. Al-activated release of ligands (such as organic acids) from the roots is a major Al tolerance mechanism in plants. In maize, Al-activated root citrate exudation plays an important role in tolerance. However, maize Al tolerance is a complex trait involving multiple genes and physiological mechanisms. Recently, transporters from the MATE family have been shown to mediate Al-activated citrate exudation in a number of plant species. Here we describe the cloning and characterization of two MATE family members in maize, ZmMATE1 and ZmMATE2, which co-localize to major Al tolerance QTL. Both genes encode plasma membrane proteins that mediate significant anion efflux when expressed in Xenopus oocytes. ZmMATE1 expression is mostly concentrated in root tissues, is up-regulated by Al and is significantly higher in Al-tolerant maize genotypes. In contrast, ZmMATE2 expression is not specifically localized to any particular tissue and does not respond to Al. [(14)C]-citrate efflux experiments in oocytes demonstrate that ZmMATE1 is a citrate transporter. In addition, ZmMATE1 expression confers a significant increase in Al tolerance in transgenic Arabidopsis. Our data suggests that ZmMATE1 is a functional homolog of the Al tolerance genes recently characterized in sorghum, barley and Arabidopsis, and is likely to underlie the largest maize Al tolerance QTL found on chromosome 6. However, ZmMATE2 most likely does not encode a citrate transporter, and could be involved in a novel Al tolerance mechanism.

Aluminum tolerance in maize is associated with higher MATE1 gene copy number

Genome structure variation, including copy number variation and presence/absence variation, comprises a large extent of maize genetic diversity; however, its effect on phenotypes remains largely unexplored. Here, we describe how copy number variation underlies a rare allele that contributes to maize aluminum (Al) tolerance. Al toxicity is the primary limitation for crop production on acid soils, which make up 50% of the world’s potentially arable lands. In a recombinant inbred line mapping population, copy number variation of the Al tolerance gene multidrug and toxic compound extrusion 1 (MATE1) is the basis for the quantitative trait locus of largest effect on phenotypic variation. This expansion in MATE1 copy number is associated with higher MATE1 expression, which in turn results in superior Al tolerance. The three MATE1 copies are identical and are part of a tandem triplication. Only three maize inbred lines carrying the three-copy allele were identified from maize and teosinte diversity panels, indicating that copy number variation for MATE1 is a rare, and quite likely recent, event. These maize lines with higher MATE1 copy number are also Al-tolerant, have high MATE1 expression, and originate from regions of highly acidic soils. Our findings show a role for copy number variation in the adaptation of maize to acidic soils in the tropics and suggest that genome structural changes may be a rapid evolutionary response to new environments.

Characterization of AtALMT1 Expression in Aluminum-Inducible Malate Release and Its Role for Rhizotoxic Stress Tolerance in Arabidopsis

Malate transporters play a critical role in aluminum (Al) tolerance responses for some plant species, such as Arabidopsis (Arabidopsis thaliana). Here, we further characterize AtALMT1, an Arabidopsis aluminum-activated malate transporter, to clarify its specific role in malate release and Al stress responses. Malate excretion from the roots of accession Columbia was sharply induced by Al, which is concomitant with the induction of AtALMT1 gene expression. The malate release was specific for Al among rhizotoxic stressors, namely cadmium, copper, erbium, lanthanum, sodium, and low pH, which accounts for the specific sensitivity of a null mutant to Al stress. Al-specific malate excretion can be explained by a combined regulation of AtALMT1 expression and activation of AtALMT1 protein, which is specific for Al. Although low pH treatment slightly induced gene expression, other treatments did not. In addition, malate excretion in Al-activated seedlings was rapidly stopped by removing Al from the solution. Other rhizotoxic stressors were not effective in maintaining malate release. Protein kinase and phosphatase inhibitor studies indicated that reversible phosphorylation was important for the transcriptional and posttranslational regulation of AtALMT1. AtALMT1 promoter-β-glucuronidase fusion lines revealed that AtALMT1 has restricted expression within the root, such that unnecessary carbon loss is likely minimized. Lastly, a natural nonsense mutation allele of AtALMT1 was identified from the Al-hypersensitive natural accession Warschau-1.

Transcriptional profiling of aluminum toxicity and tolerance responses in maize roots.

Aluminum (Al) toxicity is a major factor limiting crop yields on acid soils. There is considerable genotypic variation for Al tolerance in most common plant species. In maize (Zea mays), Al tolerance is a complex phenomenon involving multiple genes and physiological mechanisms yet uncharacterized. To begin elucidating the molecular basis of maize Al toxicity and tolerance, a detailed temporal analysis of root gene expression under Al stress was performed using microarrays with Al-tolerant and Al-sensitive genotypes. Al altered the expression of significantly more genes in the Al-sensitive genotype, presumably as a result of more severe Al toxicity. Nevertheless, several Al-regulated genes exhibited higher expression in the Al-tolerant genotype. Cell wall-related genes, as well as low phosphate-responsive genes, were found to be regulated by Al. In addition, the expression patterns of genes related to Al-activated citrate release indicated that in maize this mechanism is probably regulated by the expression level and/or function of the citrate transporter. This study is the first comprehensive survey of global transcriptional regulation under Al stress. The results described here will help to further our understanding of how mechanisms of Al toxicity and tolerance in maize are regulated at the transcriptional level.

Whole genome de novo assemblies of three divergent strains of rice, Oryza sativa, document novel gene space of aus and indica

BackgroundThe use of high throughput genome-sequencing technologies has uncovered a large extent of structural variation in eukaryotic genomes that makes important contributions to genomic diversity and phenotypic variation. When the genomes of different strains of a given organism are compared, whole genome resequencing data are typically aligned to an established reference sequence. However, when the reference differs in significant structural ways from the individuals under study, the analysis is often incomplete or inaccurate.ResultsHere, we use rice as a model to demonstrate how improvements in sequencing and assembly technology allow rapid and inexpensive de novo assembly of next generation sequence data into high-quality assemblies that can be directly compared using whole genome alignment to provide an unbiased assessment. Using this approach, we are able to accurately assess the ‘pan-genome’ of three divergent rice varieties and document several megabases of each genome absent in the other two.ConclusionsMany of the genome-specific loci are annotated to contain genes, reflecting the potential for new biological properties that would be missed by standard reference-mapping approaches. We further provide a detailed analysis of several loci associated with agriculturally important traits, including the S5 hybrid sterility locus, the Sub1 submergence tolerance locus, the LRK gene cluster associated with improved yield, and the Pup1 cluster associated with phosphorus deficiency, illustrating the utility of our approach for biological discovery. All of the data and software are openly available to support further breeding and functional studies of rice and other species.

Maize ZmALMT2 is a root anion transporter that mediates constitutive root malate efflux

Root efflux of organic acid anions underlies a major mechanism of plant aluminium (Al) tolerance on acid soils. This efflux is mediated by transporters of the Al-activated malate transporter (ALMT) or the multi-drug and toxin extrusion (MATE) families. ZmALMT2 was previously suggested to be involved in Al tolerance based on joint association-linkage mapping for maize Al tolerance. In the current study, we functionally characterized ZmALMT2 by heterologously expressing it in Xenopus laevis oocytes and transgenic Arabidopsis. In oocytes, ZmALMT2 mediated an Al-independent electrogenic transport product of organic and inorganic anion efflux. Ectopic overexpression of ZmALMT2 in an Al-hypersensitive Arabidopsis KO/KD line lacking the Al tolerance genes, AtALMT1 and AtMATE, resulted in Al-independent constitutive root malate efflux which partially restored the Al tolerance phenotype. The lack of correlation between ZmALMT2 expression and Al tolerance (e.g., expression not localized to the root tip, not up-regulated by Al, and higher in sensitive versus tolerance maize lines) also led us to question ZmALMT2s role in Al tolerance. The functional properties of the ZmALMT2 transporter presented here, along with the gene expression data, suggest that ZmALMT2 is not involved in maize Al tolerance but, rather, may play a role in mineral nutrient acquisition and transport.

Glutathione S-transferase and aluminum toxicity in maize

Aluminum (Al) toxicity induces changes in the expression of several genes, some of which are involved in plant responses to oxidative stress. Using mRNA differential display, we identified a maize Al-inducible cDNA encoding a glutathione S-transferase (GST). The gene was named GST27.2 owing to its homology to the maize gene GST27, which is known to be induced by xenobiotics. GST27.2 is present in the maize genome as a single copy and analysis of its expression pattern revealed that the gene is expressed mainly in the root tip. Expression was up-regulated in response to various Al and Cd concentrations in both Al-tolerant and Al-sensitive maize lines. Consistent with its role in plants, phylogenetic analysis of theta-type GSTs revealed that GST27.2 belongs to a group of proteins that respond to different stresses. Finally, structural analysis of the polypeptide chain indicates that the two amino acids that differ between GST27.2 and GST27 (E102K and P123L) could be responsible for alterations in activity and / or specificity. Together, these results suggest that GST27.2 may play an important part in plant defenses against Al toxicity.

Incomplete transfer of accessory loci influencing SbMATE expression underlies genetic background effects for aluminum tolerance in sorghum

Impaired root development caused by aluminum (Al) toxicity is a major cause of grain yield reduction in crops cultivated on acid soils, which are widespread worldwide. In sorghum, the major Al-tolerance locus, AltSB , is due to the function of SbMATE, which is an Al-activated root citrate transporter. Here we performed a molecular and physiological characterization of various AltSB donors and near-isogenic lines harboring various AltSB alleles. We observed a partial transfer of Al tolerance from the parents to the near-isogenic lines that was consistent across donor alleles, emphasizing the occurrence of strong genetic background effects related to AltSB . This reduction in tolerance was variable, with a 20% reduction being observed when highly Al-tolerant lines were the AltSB donors, and a reduction as great as 70% when other AltSB alleles were introgressed. This reduction in Al tolerance was closely correlated with a reduction in SbMATE expression in near-isogenic lines, suggesting incomplete transfer of loci acting in trans on SbMATE. Nevertheless, AltSB alleles from the highly Al-tolerant sources SC283 and SC566 were found to retain high SbMATE expression, presumably via elements present within or near the AltSB locus, resulting in significant transfer of the Al-tolerance phenotype to the derived near-isogenic lines. Allelic effects could not be explained by coding region polymorphisms, although occasional mutations may affect Al tolerance. Finally, we report on the extensive occurrence of alternative splicing for SbMATE, which may be an important component regulating SbMATE expression in sorghum by means of the nonsense-mediated RNA decay pathway.

Genetic dissection of Al tolerance QTLs in the maize genome by high density SNP scan

BackgroundAluminum (Al) toxicity is an important limitation to food security in tropical and subtropical regions. High Al saturation on acid soils limits root development, reducing water and nutrient uptake. In addition to naturally occurring acid soils, agricultural practices may decrease soil pH, leading to yield losses due to Al toxicity. Elucidating the genetic and molecular mechanisms underlying maize Al tolerance is expected to accelerate the development of Al-tolerant cultivars.ResultsFive genomic regions were significantly associated with Al tolerance, using 54,455 SNP markers in a recombinant inbred line population derived from Cateto Al237. Candidate genes co-localized with Al tolerance QTLs were further investigated. Near-isogenic lines (NILs) developed for ZmMATE2 were as Al-sensitive as the recurrent line, indicating that this candidate gene was not responsible for the Al tolerance QTL on chromosome 5, qALT5. However, ZmNrat1, a maize homolog to OsNrat1, which encodes an Al3+ specific transporter previously implicated in rice Al tolerance, was mapped at ~40 Mbp from qALT5. We demonstrate for the first time that ZmNrat1 is preferentially expressed in maize root tips and is up-regulated by Al, similarly to OsNrat1 in rice, suggesting a role of this gene in maize Al tolerance. The strongest-effect QTL was mapped on chromosome 6 (qALT6), within a 0.5 Mbp region where three copies of the Al tolerance gene, ZmMATE1, were found in tandem configuration. qALT6 was shown to increase Al tolerance in maize; the qALT6-NILs carrying three copies of ZmMATE1 exhibited a two-fold increase in Al tolerance, and higher expression of ZmMATE1 compared to the Al sensitive recurrent parent. Interestingly, a new source of Al tolerance via ZmMATE1 was identified in a Brazilian elite line that showed high expression of ZmMATE1 but carries a single copy of ZmMATE1.ConclusionsHigh ZmMATE1 expression, controlled either by three copies of the target gene or by an unknown molecular mechanism, is responsible for Al tolerance mediated by qALT6. As Al tolerant alleles at qALT6 are rare in maize, marker-assisted introgression of this QTL is an important strategy to improve maize adaptation to acid soils worldwide.

Redefining ‘stress resistance genes’, and why it matters

Lyza G. Maron*, Miguel A. Piñeros, Leon V. Kochian and Susan R. McCouch* 1 School of Integrative Plant Science, Plant Breeding and Genetics section, Cornell University, Ithaca, NY 14853, USA 2 Robert W. Holley Center for Agriculture and Health, USDA-ARS, Cornell University, Ithaca, NY 14853, USA Current address: Global Institute for Food Security, University of Saskatchewan, Saskatoon, S7N 5A8, Canada *Correspondence: lyza.maron@cornell.edu or srm4@cornell.edu