Plant Soil (2012) 351:121–134 DOI 10.1007/s11104-011-0937-1
REGULAR ARTICLE
Aluminum resistance mechanisms in oat (Avena sativa L.) Lorien Radmer & Mesfin Tesfaye & David A. Somers & Stephen J. Temple & Carroll P. Vance & Deborah A. Samac
Received: 11 April 2011 / Accepted: 24 July 2011 / Published online: 12 August 2011 # Springer Science+Business Media B.V. (outside the USA) 2011
Abstract Background and aims Enhanced aluminum (Al) resistance has been observed in dicots over-expressing enzymes involved in organic acid synthesis; however, this approach for improving Al resistance has not been investigated in monocots. Among the cereals, oat (Avena sativa L.) is considered to be Al resistant, but the basis of resistance is not known. Methods A hydroponic assay and hematoxylin staining for Al accumulation in roots were used to evaluate Al resistance in 15 oat cultivars. Malate and citrate release from roots was measured over a 24 h period. A malate dehydrogenase gene, neMDH, from alfalfa (Medicago sativa L.) was used to transform oat.
Results Oat seedlings were highly resistant to Al, as a concentration of 325 μM AlK(SO4)2 was needed to cause a 50% decrease in root growth. Most oat cultivars tested are naturally resistant to high concentrations of Al and effectively excluded Al from roots. Al-dependent release of malate and Al-independent release of citrate was observed. Al resistance was enhanced in a transgenic oat line with the highest accumulation of neMDH protein. However, overall root growth of this line was reduced and expression of neMDH in transgenic oat did not enhance malate secretion. Conclusions Release of malate from oat roots was associated with Al resistance, which suggests that
Responsible Editor: Hans Lambers. L. Radmer : M. Tesfaye Department of Plant Biology, University of Minnesota, 1445 Gortner Ave., St. Paul, MN 55108, USA D. A. Somers : S. J. Temple Department of Agronomy and Plant Genetics, University of Minnesota, 1991 Upper Buford Circle, St. Paul, MN 55108, USA C. P. Vance USDA-ARS-Plant Science Research Unit and Department of Agronomy and Plant Genetics, University of Minnesota, 1991 Upper Buford Circle, St. Paul, MN 55108, USA
D. A. Samac (*) USDA-ARS-Plant Science Research Unit and Department of Plant Pathology, University of Minnesota, 1991 Upper Buford Circle, St. Paul, MN 55108, USA e-mail:
[email protected] Present Address: D. A. Somers Monsanto Company, 700 Chesterfield Village Parkway, Chesterfield, MO 63017, USA Present Address: S. J. Temple Forage Genetics International, N5292 S. Gills Coulee Road, West Salem, WI 54669, USA
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malate plays a role in Al resistance of oat. Overexpression of alfalfa neMDH enhanced Al resistance in some lines but was not effective alone for crop improvement. Keywords Aluminium . Aluminum resistance . Avena sativa . Citrate . Malate dehydrogenase . Malate secretion . Oat . Sugarcane bacilliform badnavirus (ScBV) promoter
Introduction Aluminum (Al) is widespread in agricultural soils and Al toxicity is a major limiting factor in crop production throughout the world (von Uexküll and Mutert 1995). In soils with a pH of 5.5 and below, Al becomes soluble and forms phytotoxic cations, Al(H2O)63+ (also known as Al3+) and Al(OH)2+, that are rapidly taken up by plant roots (Kinraide 1991, 1997). An estimated 30– 50% of the world’s potentially arable land is acidic and current agricultural practices, along with acid rain, are causing a rise in the rate of soil acidification (Tarkalson et al. 2006; von Uexküll and Mutert 1995). Aluminum uptake by plant roots results in stunted root growth causing a decrease in the plant’s ability to absorb water and nutrients from the soil (Andersson 1988). Additional mineral ion toxicities, particularly Mn, also negatively affect plant growth in acidic soil. At low pH, many nutrients essential to plant growth are not readily available, particularly P, but also Ca, Mg, and to some extent N and K (Lambers et al. 1998). Alleviating Al toxicity in plants will not necessarily solve these problems but will moderate a major inhibiting factor for plant growth in acidic soil. Identifying Al resistant plants and understanding the mechanisms underlying resistance has been a major focus of research in many laboratories (Kochian et al. 2005). Resistance to Al has been found to occur when Al is excluded from the root apex, the main target of Al toxicity. Additionally, some plants have been shown to have mechanisms that allow them to tolerate Al in the symplasm. A predominant mechanism of Al resistance identified in cereal crops is secretion of organic acids, which chelate Al outside the root and prevent it from being taken up by root cells (Kochian et al. 2005; Ryan and Delhaize 2010). In Al resistant wheat (Triticum aestivum L.) seedlings, Al treatment rapidly stimu-
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lates secretion of malate and succinate (Delhaize et al. 1993a). The majority of malate efflux in resistant seedlings is from the root apex, although high amounts of malate accumulate behind the root tip (Ryan et al. 1995). Genetic analysis of Al resistance in wheat found that the majority of resistance is conditioned by the Alt1 locus. A gene at this locus, TaALMT1 (Al-activated malate transporter 1), encodes a malate transporter that is localized in the plasma membrane and mediates Al-activated release of malate from resistant wheat roots (Sasaki et al. 2004). Expression of this gene in barley (Hordeum vulgare L.) greatly enhances malate secretion from roots upon exposure to Al and increases Al resistance in both hydroponic and acid soil culture (Delhaize et al. 2004). More recently, Pereira et al. (2010) transformed an aluminum-sensitive wheat cultivar with TaALMT1 and found that resulting T2 lines showed increased Al3+ resistance. Al-induced secretion of citrate has been associated with Al resistance in rice (Oryza sativa L.), maize (Zea mays L.), sorghum (Sorghum bicolor (L.) Moench), and rye (Secale cereale L.) (Kochian et al. 2005). Oat (Avena sativa L.) is considered to be more resistant to Al than many other cereal crops including wheat, maize, and barley (Nava et al. 2006). However, only a few oat cultivars have been characterized specifically for their response to Al stress resulting in a lack of knowledge of the extent of Al resistance in oat. In one study, oat cultivars exhibited a range of tolerance to acidic Al-containing soil, which was assumed to reflect their relative Al resistance (Foy et al. 1987). In solution culture, six oat cultivars were shown to vary in Al tolerance (Wheeler et al. 1992). Inheritance studies conducted with Brazilian Al resistant cultivated oat genotypes found resistance to be conditioned by single genes (Castilhos et al. 2011; Nava et al. 2006). Zheng et al. (1998) found that oat plants release malate and citrate after exposure to 50 μM Al. The amount of organic acids secreted was low compared to the amount of organic acids produced by Al resistant wheat, which led the authors to suggest that additional resistance mechanisms may be present in oat. In the diploid oat, A. strigosa Schreb., four QTLs for Al tolerance were identified (Wight et al. 2006). Since oat is expected to have highly effective mechanisms of resistance or tolerance, further investigation of the response of oat to Al stress is warranted.
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Overexpression of genes encoding enzymes involved in organic acid production in transgenic plants has been shown to enhance Al tolerance. In alfalfa (Medicago sativa L.), overexpression of a novel form of malate dehydrogenase (neMDH) resulted in an increase in malate concentration in roots, release of greater amounts of malate from roots, and enhanced Al tolerance compared to nontransgenic control plants (Tesfaye et al. 2001). neMDH is normally expressed in alfalfa root nodules and has a much higher specificity for oxaloacetate and NADH than other MDH isoforms, which drives the reaction towards malate production (Miller et al. 1998). Likewise, overexpression of citrate synthase was reported to increase Al tolerance in Arabidopsis (Koyama et al. 2000), Brassica napus L. (Anoop et al. 2003), and alfalfa (Barone et al. 2008). Whether overexpression of genes involved in organic acid synthesis will enhance Al resistance in a cereal crop has not been tested. The objectives of this research were to develop screening methods for identifying Al tolerance or resistance mechanisms in oat and to test if malate release can be enhanced by gene overexpression. In this research, oat (cv. Belle) embryos were transformed, using the neMDH gene controlled by the Sugarcane bacilliform badnavirus (ScBV) promoter. In oat, the ScBV promoter conveys constitutive gene expression in most tissues of the plant with the highest expression in the vascular tissue (Al-Saady et al. 2004; Tzafrir et al. 1998). Plants were assayed for Al tolerance in a hydroponic system and by hematoxylin staining to evaluate Al exclusion from root tips. Using the same methods, 15 oat cultivars were evaluated for root growth, relative Al tolerance, and Al exclusion. In contrast to previously published reports we found that oat seedlings secreted large amounts of malate when grown in hydroponic culture containing a high Al concentration and that exudation correlated with Al resistance.
Materials and methods Plant material Oat cultivars Baker, Belle, Drumlin, Esker, Kame, Leonard, Moraine, Morton, Reeves, Richard, Riser, Sesqui, Spurs, Wabasha, and Winona were tested for Al tolerance. These were the most widely grown
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cultivars in the Midwestern U.S. at the time of the experiment. Seed was obtained from Dr. Deon Stuthman, Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN. Hydroponic assay of Al tolerance Seed was vernalized for 24 h at 4°C in Petri plates between moist pieces of filter paper. Plates were then moved to a 30°C incubator for 24 h. Germinated seeds with roots of approximately equal length were placed individually in a 48 mm plastic cup with a nylon mesh bottom with 3 mm openings. Hydroponic chambers were constructed using 15 L plastic containers (ClearView Storage Boxes, Sterilite, Townsend, MA) fitted with a plexiglass insert with holes to accommodate 12 plastic cups. The cups were suspended so that the mesh touched the surface of the assay solution. The chambers were filled with 7.3 L of preconditioning solution consisting of 0.4 mM CaCl2 adjusted to pH 4.4 using 1M HCl. Seedlings were grown for 24 h in the preconditioning solution under constant aeration with ambient room light and then the length of the three longest roots of each seedling was measured using a mm ruler. Seedlings were then moved to chambers that contained fresh control solution, 0.4 mM CaCl2 at pH 4.4, or an experimental solution containing 0.4 mM CaCl2 and different amounts of AlK(SO4)2 at pH 4.4. The experimental and control solutions were adjusted to pH 4.4 using 1M HCl. After 24 h under constant aeration in control and test solutions, the length of the three longest roots was measured. The average root length was calculated for each plant. The relative root growth ratio (RRGR) for each Al concentration was calculated using: RAf RAi RRGR ¼ RCf RCi in which RAf was the final aluminum-grown root length, RAi the initial aluminum-grown root length, RCf the final control root length, and RCi the initial control root length. Sixteen seeds were germinated from each transgenic and control line and seedlings placed in the preconditioning treatment as described above. Half of the plants from each line were then moved to a hydroponic chamber containing an experimental
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solution: 0.4 mM CaCl2, 325 μM AlK(SO4)2, pH 4.4. The remaining seedlings were placed in fresh preconditioning solution. The length of the three longest roots was measured. After 24 h under ambient lab conditions and constant aeration, the length of the three longest roots was measured a second time. This experiment was done three times and RRGR was calculated using the mean values of each line averaged across seedlings in each biological replicate. Statistical analysis of RRGR was performed using the General Linear Model procedure in SAS (SAS Institute 2003). Twenty-five seeds of each of 15 cultivars (Baker, Belle, Drumlin, Esker, Kame, Leonard, Moraine, Morton, Reeves, Richard, Riser, Sesqui, Spurs, Wabasha, and Winona) were vernalized, germinated, and grown in preconditioning solution as described above. The 18 seedlings with the longest roots were chosen. Nine seedlings were placed in a control solution of 0.4 mM CaCl2 pH 4.4 and another nine seedlings were placed in a 325 μM AlK(SO4)2, 0.4 mM CaCl2, pH 4.4 solution. Cups containing seedlings were randomized before being placed in the hydroponic chambers. The three longest roots of each seedling were measured and seedlings were incubated in hydroponic chambers for 24 h under ambient lab conditions and constant aeration. After a further 24 h growth, the three longest roots of each seedling were measured again and the relative root growth ratio for the cultivar was calculated as described above. At the end of the Al experiment, the roots of the seedlings were stained with hematoxylin as described below. The experiment was done three times and data were analyzed as described above. Detection of Al uptake in oat roots To detect Al uptake in Al-treated and control plants, roots were rinsed in distilled water for 30 min to remove any nutrient growth solution, and then stained with a solution of 0.1% hematoxylin (Sigma-Aldrich, St. Louis, MO) and 0.01% KI for 30 min (Canaado et al. 1999). Excess hematoxylin stain was washed off with a 30 min rinse in distilled water. Finally, roots were examined with a dissecting microscope and photographed. To examine Al within roots, seedlings were grown in 0 mM Al or 325 μM Al, and then stained with 100 μM morin (Sigma-Aldrich) (Larsen et al. 1996).
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Approximately 1 cm of root tip was excised from each seedling, embedded in 3% agarose, and 50 μm cross-sections were made from approximately 0.5– 1 mm from the root tip using a vibratome. Sections were placed on a microscope slide and examined with an Olympus IX70 inverted microscope by bright field and epifluorescence (440 nm excitation and 510 nm emission). Measuring malate and citrate exudation To determine the amount of malate exuded from root tips of seedlings of cv. Belle, 10 germinating seeds were placed in a 35 mm plastic cup with a 3 mm nylon mesh bottom in a 50 mL sterile conical polypropylene tube (Corning Incorporated, Corning, NY). Seedlings were grown in 50 mL preconditioning solution (0.4 mM CaCl2, pH 4.4), with constant shaking at 100 rpm for 24 h. The preconditioning solution was replaced with 50 mL of 0.4 mM CaCl2 with 0 mM, 100 μM, 200 μM, or 300 μM AlK(SO4)2 at pH 4.4. Seedlings were grown for an additional 24 h with constant shaking at 100 rpm. In a separate study, malate and citrate exudates from seedlings of four additional cultivars showing a range of Al resistance were collected in the same way using 325 μM AlK(SO4)2 at pH 4.4. The solutions were frozen at −80°C, lyophilized, and material dissolved in distilled water. Aliquots of the concentrated samples were used for analysis of malate using an L-malic acid enzymatic assay (Megazyme, Wicklow, Ireland). Cations were removed from samples using Empore chelating disks (3 M Company, Eagan, MN) before measuring citrate content with the Megazyme citric acid enzymatic assay. Three to four root exudate samples were measured for each treatment and the experiment was done twice. Oat transformation and characterization of transgenic lines The plasmid pScBV3-MDH11 used for transformation consisted of a full-length cDNA of neMDH from alfalfa (Miller et al. 1998) inserted behind the ScBV promoter in pRT106’-pSCBV3-BB (Tzafrir et al. 1998). Embyogenic tissue cultures derived from mature embryos of the oat cv. Belle were cobombarded with pScBV3-MDH11 and pH24, a plasmid
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with a paromomycin selectable marker (Fromm et al. 1986), as described previously (Torbert et al. 1998). Seed from paromomycin resistant regenerated T0 plants were grown in the greenhouse and seed collected from individual self-pollinated plants. Forty-two T2 lines were assayed as follows to identify lines putatively homozygous for the neMDH transgene. RNA was extracted from seedling roots using the RNeasy kit (Qiagen, Valencia, CA). Reverse transcription- (RT) PCR was performed using the Access RT-PCR System (Promega Corp., Madison, WI) with the primers M11F 5′-GACCTGCATCTC TATGATATCG-3′ and MRTR 5′-CAACAACTGGAA CATCCACATC-3′ corresponding to positions 384– 405 and 801–822 of the neMDH cDNA sequence (Genbank accession AF020273). The PCR amplification conditions were: 1 cycle of 48°C for 45 min, 1 cycle of 94°C for 2 min, followed by 40 cycles of 94° C for 30 s, 53°C for 30 s, and 68°C for 1 min, with a final cycle of 68°C for 7 min. Products were separated on 1% agarose gels and stained with ethidium bromide. Lines in which all eight seedlings tested produced mRNA of the transgene were saved for further testing. Where possible, sister lines from the same T0 plant but in which the transgene was not expressed were identified using the same technique. To estimate transgene copy number, Southern blot analysis was done using genomic DNA digested with Xba1, which has a single site in pScBV3-MDH11. Blots were probed with a 1.65 kbp fragment of neMDH as described previously (Tesfaye et al. 2001). Accumulation of neMDH protein in oat roots was detected by immunoblotting as described previously (Tesfaye et al. 2001) using protein extracted from plant roots grown hydroponically in a 0.4 mM CaCl2 solution. Malate and citrate concentration in root exudates were measured as described above.
Results Response of seedlings from the cultivar Belle to Al treatment When cv. Belle seedlings were grown in increasing Al concentrations the relative root ratio decreased in a concentration-dependent manner, indicating that there was greater root growth inhibition at the higher Al
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concentrations (Fig. 1a). However, inhibition of root growth was similar at 250, 300, and 350 μM AlK (SO4)2. These concentrations result in 80 μM, 90 μM and 98 μM free Al activity, respectively, as determined by GEOCHEM-EZ (Shaff et al. 2010). It was not possible to test higher concentrations of Al because additional Al decreased the assay solution below pH 4.4. Addition of base to raise the pH would alter the composition of Al ions in the solution. The Al concentration at which cv. Belle plants reproducibly displayed approximately 50% root growth inhibition under hydroponic conditions was 325 μM AlK (SO4)2 in 0.4 mM CaCl2, pH 4.4, which results in 94 μM free Al activity. This concentration was used for further comparative tests. Hematoxylin is an Al-specific dye that has a purple color when it forms a complex with Al (Polle et al. 1978). After exposure to Al for 24 h, root tips of cv. Belle plants had low to moderate amounts of staining with hematoxylin (Fig. 1b). An increase in staining occurred with increasing Al concentration. Most staining occurred in epidermal cells, the root tip, and material loosely associated with the roots with relatively little stain associated with root cortical cells. Roots grown in the control solution did not stain. Little distortion or growth abnormalities of the root tips were observed, even at the highest Al concentration. Cross sections of root tips exposed to 325 μM AlK (SO4)2 for 24 h were made and stained with morin to identify locations in the root were Al accumulated (Fig. 2). There was a small amount of background fluorescence across the root sections in roots grown in the control solution after morin staining (Fig. 2b). A few root border cells and other cells detached from the root tip fluoresced brightly, without exposure to Al. Roots exposed to Al displayed a moderate number of brightly fluorescing cells (Fig. 2d). Fluorescence was observed in distinct patches of cells in the outer cell layer and several underlying layers of cortical cells. Fluorescence was often associated with damaged cells visible by light microscopy (Fig. 2c). A greater number of root border cells fluoresced after Al exposure than in the control treatment. Malate exudation was examined in roots of cv. Belle seedlings at three Al concentrations. The amount of malate in root exudates increased with Al concentration (Fig. 3). The Al treatments increased malate in exudates 22- to 41-fold, from an average of
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Fig. 1 Response of cv. Belle seedlings to Al-containing hydroponic solution culture. a The relative root growth ratio of cv. Belle seedlings. Growth in Al treatment solutions (pH 4.4) was compared to growth in the 0.4 mM CaCl2 pH 4.4 control solution
over 24 h. Error bars indicate standard error. b Cultivar Belle root tips grown in a range of Al concentrations and stained with hematoxylin. Scale bar indicates 1 mm
1.24 nmol seedling−1 in the control treatment of 0 μM Al to 27.8 nmol seedling−1 in the 100 μM AlK(SO4)2 treatment and 51 nmol seedling−1 in the 300 μM AlK (SO4)2 treatment.
plants from ‘Belle’ and ‘Sesqui’ were not significantly different (p
