Journal of Experimental Botany, Vol. 54, No. 393, pp. 2745-2756,
December 1, 2003
© 2003 Oxford University Press
Comparison of Al-induced gene expression in sensitive and tolerant soybean cultivars
Received 11 August 2003; Accepted 2 September 2003
Institute of Plant Genetics and Crop Plant Research (IPK), 3 Corrensstrasse, D-06466 Gatersleben, Germany
* Present address and to whom correspondence should be sent: Emma-Noether Group, AG Flechsig, Institute of Virology and Immunology, University of Wuerzburg, 7 Versbacher st., D-97078, Wuerzburg, Germany. Fax: +49 (0)931 201 49553. E-mail: ermolavo{at}yahoo.de
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Abstract |
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In order to identify genes involved in soybean resistance to aluminium (Al) stress differential gene expression patterns of Al-stressed and non-stressed tolerant and sensitive soybean cultivars were compared. Out of eight described genes, potentially related to mechanisms of aluminium stress, only phosphoenolpyruvate carboxylase (PEPC) revealed enhanced expression in roots of tolerant as compared to sensitive soybean cultivars under stress conditions. Additionally, two novel full-length cDNA sequences, homologous to translationally controlled tumour proteins (TCTP, clone 58, GenBank accession number AF421558 [GenBank] ) and inosine-5'-monophosphate dehydrogenases (IMPDH, clone 633, GenBank accession number AF421559 [GenBank] ) with enhanced expression of the corresponding genes only in roots of Al-tolerant soybean cultivar under stress conditions were isolated and characterized. For functional analysis full-length cDNA 633 was transferred in Arabidopsis thaliana. Only 6% of the seedlings from the wild type survived Al stress, whereas 86% of transgenics were vital demonstrating superiority in stress protection. Compared with the wild type, transgenic plants showed diminished Al penetration into the roots after the stress treatment especially in the division and elongation zones of the roots. Formation of numerous lateral roots in transgenic plants with low elicited callose accumulation under stress conditions indicated ability of the IMPDH homologue to mediate aluminium tolerance in transgenic plants. Possible functional activities of Al up-regulated genes in resistance mechanisms are discussed.
Key words: Aluminium resistance, aluminium stress, Differential Display, gene expression, soybean.
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Introduction |
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Al does not exert any known function in plant metabolism and belongs to the non-essential metals. Under neutral soil conditions, it exists in the non-phytotoxic insoluble form, whereas acidification of soil and soil water below pH 4.5 dramatically enhances release of the phytotoxic aluminium ion (MacDonald and Martin, 1988; Foy, 1984). Since acid soils occupy up to 40% of the world’s arable land (Kochian, 1995), aluminium phytotoxicity may be considered as one of the major limiting factors of crop productivity in the world (Foy et al., 1978; Matsumoto, 2000).
It is widely accepted, that the terminal 2–3 mm of roots including meristem and root cap play a major role in aluminium toxicity and tolerance (Ryan et al., 1992; de la Fuente et al., 1997; de la Fuente-Martinez et al., 1999; Kollmeier et al., 2000). Al-stressed roots become thick and brown and inefficient in water and nutrient uptake (Rengel, 1992). Al inhibits both cell division and cell elongation in root tips, whereas the mature parts of the root remain unaffected (Ryan et al., 1993; Kochian, 1995). The fast response of root tip cells to Al suggests inhibition of cell elongation, rather than division, since one round of the cell cycle in each cell of the roots takes approximately 24 h (Kochian, 1995). In addition, phytotoxic Al very rapidly induces callose biosynthesis in root cells (Zhang et al., 1994; Horst, 1995; Kataoka and Nakamishi, 2001). Al-elicited callose accumulation correlates temporarily with the inhibition of root elongation, and species tolerant to Al show less Al-induced callose accumulation compared with the sensitive ones (Horst et al., 1997).
High genetic variability of Al tolerance (Ma et al., 2001; Delhaize et al., 2001) indicates that some plant species evolved special mechanisms to survive stress conditions. Strategies of plant resistance include the exclusion of Al penetration into root cells (apoplastic mechanisms) and/or neutralization of toxic Al within the cell (symplastic mechanisms). Organic acid exudation by root cells (Delhaize et al., 1993; Miyasaka et al., 1991; Pellet et al., 1995), apoplastic binding of Al ions in the cell wall (Kochian, 1995; Kollmeier et al., 2001) and lowering of the rhizosphere pH (Miyasaka et al., 1989) are the best investigated apoplastic mechanisms of plant resistance. The correlation between organic acid exudation and Al resistance was initially detected by Miyasaka et al. (1991) in snap bean and found to play an important role in many other plants (Ryan et al., 1995; Yang et al., 2000). Symplastic mechanisms of plant response to Al stress are still poorly understood. Examples of the resistance mechanisms inside the plant cell include the internal chelation of toxic Al by oxalic acid in buckwheat (Ma et al., 1997).
At present, it is not clear whether the induced expression of genes involved in apoplastic or symplastic resistance mechanisms or the activation of pre-existing systems on the protein level or both are responsible for the acquisition of stress tolerance. Several genes up-regulated under conditions of Al phytotoxicity have been discovered. The genes corresponding to wali1–wali5 cDNA fragments were induced very rapidly by Al stress (Snowden and Gardner, 1993). These fragments, as well as wali6 and wali7 (Richards et al., 1994), were discovered by screening of a cDNA library from stressed wheat root tips. The genes homologous to glutathione-S-transferase (GST) and phenylalanine ammonia lyase (PAL genes) were induced by Al stress and Pi starvation in tobacco (Ezaki et al., 1995). The following genes were shown as up-regulated: Sali3-2 and Sali5-4 in soybean (Rhagland and Soliman, 1997), ZmaI in maize (Menossi et al., 1999), Mtn29 in Barrel medic (Gamas et al., 1996), pEARLI8 and pEARLI1, pEARLI2, pEARLI4, and pEARLI5 in Arabidopsis thaliana (Richards et al., 1998) and phosphatidylserine synthase in wheat (Delhaize et al., 1999). The majority of the genes up-regulated under stress conditions belong to the general stress response as, for instance, the genes encoding the metallothionein-like protein wali1 (Snowden and Gardner, 1993) or genes of unknown function such as wali7 (Richards et al., 1994) or ZmaI (Menossi et al., 1999). In order to prove the function of those genes in stress resistance, some of them were over-expressed in Arabidopsis thaliana plants (Ezaki et al., 2000, 2001). Only the Arabidopsis gene encoding the blue copper binding protein (AtBCB) and the tobacco genes coding for gluthathione-S-transferase (parB), peroxidase (NtPox) and GDP-dissociation inhibitor (NtGDI1) conferred some resistance to Al under moderate Al stress conditions. Upon higher Al concentration, however, the transgenic plants were not superior to the controls in surviving stress.
As far as is known, no comparative analyses of gene expression in stressed and non-stressed Al-tolerant and -sensitive plants have been performed. In the present paper, northern blotting was used to visualize expression patterns of several genes known to be potentially involved in plant Al-resistance. The expression patterns were compared between Al-tolerant and -sensitive soybean cultivars stressed and non-stressed by Al. Furthermore, novel genes showing enhanced transcript abundance under Al stress were identified by comparing Differential Display patterns of tolerant and sensitive soybean cultivars under stress and non-stress conditions. A complete cDNA representing one of two genes up-regulated under Al stress was transferred in Arabidopsis plants. Al-stress tolerance was also measured, as well as Al penetration and Al-induced callose accumulation in roots of transgenic plants compared with the wild type.
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Plant material
Seeds of Glycine max cv. Tambora (Al-tolerant) and cv. Malabar (Al-sensitive) were obtained from the Research Institute for Food Crop Biotechnology (RIFCB), Jalan Tentara Pelajar 3A, Bogor, Indonesia. Arabidopsis thaliana cv. Columbia (GenBank, Gatersleben, Germany) was used for the production of transgenic plants.
Growth conditions and stress treatment
Soybean seeds were germinated with aeration in the dark on wet filter paper (1 mM CaCl2) for 3 d. Root tips were cut off to stimulate the growth of lateral roots and plantlets were transferred to modified Hoagland solution [5 mM Ca(NO3)2, 1 mM MgSO4, 5 mM KNO3, 1 mM KH2PO4 with micronutrients: 50 µM H3BO3, 0.3 µM CuSO4, 0.1 µM (NH4)6Mo7O24, 4.5 µM MnCl2, 3.8 µM ZnSO4] and grown with aeration in the greenhouse for the next 4 d. Subsequent Al treatment was performed at pH 4.0–4.2 in Hoagland solution either supplemented with AlCl3 to a final concentration of 300 µM (Al stress) or without AlCl3 (acidic stress) for 4, 24 or 48 h as indicated. After stress treatment, roots were washed with cold water, root tips (1–2 cm) were separated on ice, frozen in liquid nitrogen and stored at –70 °C until RNA isolation.
RNA isolation
RNA extraction was performed by the guanidine hydrochloride method (Logemann et al., 1987). RNA was dissolved in DEPC-treated water and stored at -70 °C. RNA quality and quantity have been verified by denaturing RNA agarose gel electrophoresis (Sambrook et al., 1989) and spectrophotometrically by using a Biophotometer (Eppendorf, Cologne, Germany).
Differential Display RT-PCR
Differential Display RT-PCR (DD) was performed according to Liang et al. (1993) with 11-mer primers 5'-CTTGATTGACC-3', 5'-GGATCATCTCG-3', 5'-CATCGAATGAC-3' as well as degenerated anchored primers T13NA, T13NC and T13NG. [33P]dCTP (Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany) was used in amplification reactions as the radioactive label. All PCR reactions were made twice using the same cDNA sample. Aliquots (3 µl each) of amplification products were resolved on a 6% polyacrylamide sequencing gel. After exposing the gel to the autoradiographic film, the bands of interest were cut off, eluted in TE buffer (Sambrook et al., 1989) at 37 °C for 4–5 h, reamplified and cloned into the PCR Script SK+- cloning vector using the PCR-Script Amp Cloning Kit (Stratagene, La Jolla, CA, USA).
Marathon cDNA amplification
Marathon cDNA amplification was performed according to the manufacturer’s instructions (Clontech, Palo Alto, CA, USA) with primer specific to the DD fragment TA22 (5'-GGTCGTGCTAG ATTCCCGGAAG-3'). The products of PCR reactions were resolved on a 1% agarose gel. The fragments amplified in the root tip samples from Al-treated tolerant plants, but not sensitive and non-treated plants, were taken for the further experiments.
Cloning and DNA sequence analysis
Standard methods of restriction analysis and gel-electrophoresis of nucleic acids were applied according to Sambrook et al. (1989). The NucleoSpin Extract kit (Macherey-Nagel, Düren, Germany) was used for PCR fragment isolations. Plasmid DNA was isolated using the FlexiPrep kit (Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany). Midi preparations of plasmid DNA were performed using the Plasmid Midi kit (Qiagen, Hilden, Germany). The E. coli strains XL-1 Blue, XL-10 Gold and HB101 were transformed according to Hanahan (1988). Cloning of PCR fragments was performed by the PCR-Script Amp Cloning kit according to the manufacturer’s instructions (Stratagene, La Jolla, CA, USA). DNA was sequenced according to Chen and Seeburg (1985) by automated ALF-sequencing equipment. DNAMAN Sequence Analysis Software, Version 4.1 (Lynnon BioSoft, Canada) was used for sequence analysis and editing. Search for homologous nucleic acid and protein sequences was done by BLAST (Altschul et al., 1990) at http://www.ncbi.nlm.nih.gov/blast/blast.cgi) or at http://www.ncbi.nlm.nih.gov/BLAST/ Genome/ara.html for Arabidopsis genome. Search for protein motifs and functional sites was done at http://www.isrec.isb-sib.ch/software/PFSCAN_form.html (Profile Scan server at Swiss Institute for Experimental Cancer Research) and http://www.expasy.ch/tools/scnpsit1.html (Scan Prosite ExPASy server at Swiss Institute of Bioinformatics).
Northern and reverse northern blotting
Northern blotting was performed according to Sambrook et al. (1989) using Church buffer (Church and Gilbert, 1984) with the genes coding for calreticulin, metallothioneins MT4, MT1 and metallothionein from pea, glutathione-S-transferase, 
-subunit of heterotrimeric G-protein, malate dehydrogenase and phosphoenolpyruvate carboxylase from Vicia faba. The northern blotting with transgenic plants was performed with 20 µg of root total RNA transferred on the Hybond Nylon N+ membrane and hybridized with [32P]dCTP-labelled complete cDNA or 18S rDNA probe as a loading control. Reverse northern blotting was performed according to the Clontech (Palo Alto, CA, USA) protocol for hybridization with cDNA. cDNA was synthesized by Super Script RNaseH– Reverse Transcriptase (Gibco BRL Life Technologies, Gaithesburg, MD, USA) in a final reaction volume of 80 µl. Aliquots (27 µl each) of reaction mixture were used in random primed labelling reaction performed by using the Megaprime DNA Labelling kit (Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany). [32P]dCTP and [32P]dATP (Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany) were used for radioactive labelling and the labelled probes were purified by NucTrap columns (Stratagene, La Jolla, CA, USA). Hybridizations were performed in Church buffer (Church and Gilbert, 1984) at 60 °C on membranes containing aliquots with 10 µg of purified Differential Display or Marathon PCR fragments. The hybridization results were analysed using a Phosphor Imager BAS 2000, Fujix and TINA 2.09 software (Raytest Isotopenmessgeraete GmbH, Germany).
cDNA library
The cDNA library was constructed by using the 
-ZAP Express cDNA synthesis kit (Stratagene, La Jolla, CA, USA). Aliquots containing 75 µg of both total RNA isolated from Al-stressed suspension culture of the Al-tolerant Glycine max cv. Wilis and the root tips of Al-tolerant cv. Tambora were mixed 1:1 and used for cDNA synthesis. Soybean cv. Wilis was used as better performing in the suspension culture; cv. Tambora, however, showed better Al-tolerance in nutrient solution. Gigapack III Gold Packaging Extract (Life Technologies, Karlsruhe, Germany) was used for packaging. The library was screened by purified PCR fragments (100 ng) obtained by Marathon cDNA amplification. Hybridizations were performed in Church buffer (Church and Gilbert, 1984) at 65 °C. The positive clones were subjected to in vivo excision and subsequent sequencing.
Plant transformation
The protein-encoding sequence of clone 633 was amplified by high fidelity PyroBest DNA polymerase (TaKaRa, Japan) using specific primers. The primers were designed for synthesis of the full-length protein in frame with restriction sites ‘SmaI’ and ‘NotI’ at the 5'- and 3'-end of the sequence, respectively. The PCR products were resolved on a 1% agarose gel, purified using the NucleoSpin Extract kit (Macherey-Nagel, Duren, Germany) and cloned into the plasmid pRTRA7/3 (kindly provided by S Miroshnichenko, IPK, Gatersleben) containing the Cauliflower Mosaic Virus 35S promoter in the 5'-region, c-myc-tag and terminator poly-A sequence at the 3'-end. After recombinant plasmid verification by restriction analysis, partial digestion was made using the restriction enzyme HindIII. The vector plasmid pBIN19 was also digested by HindIII and dephosphorylated to block self-ligation according to Sambrook et al. (1989). The construct consisting of the CMV 35S promoter, coding region of the gene 633, c-myc-tag, and poly-A region were cloned into the pBIN19 HindIII sites. The recombinant plasmid was transformed into Arabidopsis thaliana cv. Columbia according to Clough and Bent (1998). After selection of F1 transgenic plantlets on MS agar (Murashige and Skoog, 1962) containing 30 µg ml–1 kanamycin, resistant seedlings were transferred to soil and grown to maturation. Resistant F2 progeny were selected to investigate their stress tolerance.
Stress resistance and histological studies on transgenic plants
Evaluation of Al resistance was performed on 4–7-d-old seedlings, which were transferred to Hoagland solution, pH 5.7 (control), pH 4.0 (acidic stress), or Hoagland solution, pH 4.0, supplemented with Al chloride in the concentration range from 50–300 µM.
Histological detection of Al penetration into plant roots was performed by morin staining according to Larsen et al. (1996). Briefly, the roots of 7-d-old seedlings were treated by 200 µM Al chloride in Hoagland solution, pH 4.0 for 2 h, washed in 5 mM NH4OAc, pH 5.0 for 10 min and stained with 100 µM morin (Fluka, Deisenhofen, Germany) in 5 mM NH4OAc, pH 5.0 for 1 h. After washing in 5 mM NH4OAc, pH 5.0 for 10 min, stained roots were visualized under a fluorescence microscope (Zeiss, Axioplan II) using the filter for DAPI-FITC-Rhodamine fluorescence.
Histological detection of Al-induced callose biosynthesis in plant roots was performed by aniline blue staining according to Schmohl et al. (2000). The roots of 7-d-old seedlings were treated with 200 µM Al chloride in Hoagland solution, pH 4.0 for 48 h, cut and, after washing in tri-distilled water for 5 min, stained with aniline blue for 2–3 min followed by washing in tri-distilled water for 5 min. Roots of wild-type and transgenic plants were stained and washed simultaneously to ensure uniformity of the staining procedure. After staining the roots were visualized under the same fluorescence microscope using the same filter combination as described for the morin staining procedure.
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Transcript levels of the genes potentially related to Al stress
To elucidate the response of Al-treated roots from Al-tolerant and -sensitive soybean cultivars on mRNA level, transcript abundances were tested for several genes potentially related to Al stress. Eight functionally different genes encoding calreticulin, several metallothionein-like proteins (MT), glutathione-S-transferase (GST),
-subunit of heterotrimeric G-protein, malate dehydrogenase (MDH), and phosphoenolpyruvate carboxylase (PEPC) and up-regulated by phytotoxic Al in several plant species (Kochian, 1995; Snowden and Gardner, 1993; Ezaki et al., 1996, 2000; Li et al., 2000) were selected.
Neither the genes coding for the Ca-binding protein calreticulin, the antioxidant enzyme GST and the -subunit of a G-protein, nor the MT-like proteins were up-regulated during Al stress in roots of sensitive or tolerant cultivars (results not shown) indicating that these genes are not involved in mechanisms of stress toxicity or tolerance of soybean. The genes encoding the enzymes MDH and PEPC involved in organic acid production showed different regulation of their transcript levels during Al-stress. The gene activity of MDH was not up-regulated, whereas the transcript level of PEPC gene was increased in Al-stressed soybean roots of both tolerant cv. Tambora and sensitive cv. Malabar. The stress-induced increase of the transcript level was 5.4-fold in the tolerant cultivar. Induction of this gene expression in the stressed, sensitive cultivar was only half that of the tolerant one (2.6-fold, compared with the non-stressed, tolerant cultivar). In non-stressed roots of cv. Malabar PEPC gene expression was below the detection level (Fig. 1). Among all the genes probed, only PEPC showed enhanced expression in the tolerant cultivar suggesting that it may be involved in the Al resistance mechanism of soybean.
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Search for novel genes up-regulated by Al stress
Gene expression patterns in roots of stressed and non-stressed Al-tolerant (cv. Tambora) and -sensitive (cv. Malabar) soybean cultivars were comparatively analysed by Differential Display RT-PCR (DD) in order to search for the genes responsible for stress resistance.
It was supposed that expression of the genes specifically involved in plant resistance should be the highest in roots of the stressed, tolerant cultivar, compared with non-stressed or Al-stressed sensitive plants. cDNA fragments corresponding to the genes up-regulated in Al-treated roots of the tolerant cultivar, but not in the non-stressed or Al-stressed sensitive one, were selected. The expression of the gene containing the TA22 fragment corresponded to this model, as shown by the Differential Display pattern (Fig. 2). The expression level of the gene corresponding to cloned TA22 was also shown to be increased 7-fold compared with non-stressed controls by reverse northern blotting (Fig. 3).
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Differential Display fragments represent the short 3'-part of a mRNA beside the poly-A tail, very often from the untranslated region only (Liang et al., 1993), which makes it difficult to use them in cDNA bank screening or sequence identification. Longer cDNA fragments 1 and 2 were isolated by the Marathon cDNA amplification method using the primers designed from the TA22 fragment (Fig. 4). The expression of the genes containing cDNA fragments 1 and 2 was enhanced in stressed roots of Al-tolerant plants 208- and 16-fold, respectively, compared with non-stressed controls (Fig. 5A); 4-fold and 2-fold, compared with Al-stressed, sensitive plants (Fig. 5B).
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Novel Al-stress-induced genes: characterization of their transcripts
In order to obtain complete cDNAs corresponding to putative fragments 1 and 2 isolated by the Marathon amplification method, a
-ZAP cDNA library was created. To represent cDNAs corresponding to stress-induced genes of differentiated and non-differentiated cells in the library, Al-stressed suspension cultures of the tolerant soybean cv. Wilis and root tips of the tolerant cv. Tambora were used as RNA sources. Soybean cv. Wilis was used as better performing in the cell culture; cv. Tambora, however, showed better Al-tolerance in hydroponic culture. The complete cDNAs were isolated and designated as 58 cDNA corresponding to fragment 2 and 633 cDNA corresponding to fragment 1. Al-stress-induced expression was detected in soybean root tips for the genes represented by the cDNAs 58 and 633 by reverse northern blotting (result not shown). The two full-length cDNAs were completely sequenced. The protein encoded by cDNA 58 consists of 180 amino acids. It shows an average identity rate of 86.7% with plant translationally controlled tumour proteins (TCTPs). Analysis of the probable secondary protein structure as well as the hydropathy plot of this protein revealed one relatively long hydrophobic region at its C-terminus (amino acids 124–180), but not many potential co- or post-translational modification sites. For example, only five phosphorylation sites were found (Fig. 6). The potential TCTP signature motif 1 overlaps with one of the predicted myristoylation sites. The N-terminal topology of the myristoylation sites involving TCTP signature 1 may either point to probable membrane-association or to protein–protein interactions. TCTP signature 2 overlaps with a phosphorylation site, indicating potential regulation of TCTP activity by protein kinases. Two additional phosphorylation sites are located near the N-terminus of the protein. However, it is not known, whether all potential phosphorylation or myristoylation sites are functional in vivo.
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The protein sequence encoded by clone 633 consists of 337 amino acids showing no hydrophobic regions (Fig. 6). Two functional domains were identified, an inosine-monophosphate dehydrogenase (IMPDH, amino acids 85–278) and a flavine-mononucleotide (FMN) binding domain (amino acids 116–238). Furthermore, two potential phosphorylation sites were found (Fig. 6). One of the main features of this predicted amino acid sequence is a lot of potential myristoylation sites. There are 12 myristoylation sites, the majority of them is located in that region, where IMPDH and FMN binding domains overlap. The protein has 69% identity with Arabidopsis and 48% with human proteins, however, on the nucleotide level no significant homology has been found in plant genomes, including the Arabidopsis complete genome sequence.
IMPDH homologue increases Al resistance of transgenic plants
In order to investigate the function of Al-induced gene activity corresponding to IMPDH cDNA, the protein-coding region was cloned behind the 35S promoter of Cauliflower mosaic virus enabling ubiquitous expression in plants. The NPTII gene was inserted to allow kanamycin selection of transgenic Arabidopsis plants. The expression of transgene in kanamycin-resistant F2 plantlets was proved by northern blotting (Fig. 7).
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Wild-type Arabidopsis cv. Columbia seedlings were tested for their sensitivity to Al stress. Restricted growth of the seedlings was already observed at 50 µmol AlCl3. 5.6% Arabidopsis plantlets survived 48 h treatment by 200 µmol Al chloride, whereas at 300 µmol all the seedlings died showing intensive chlorosis (results not shown). The stress effects could be directly attributed to Al toxicity since acidic stress (pH 4.0) did not restrict the growth.
Al-tolerance of transgenic and wild-type plants was comparatively assessed at 200 µmol AlCl3 (pH 4.0). Transgenic seedlings showed significantly higher tolerance to Al stress. 80–93% of transgenics survived 48 h stress treatment, whereas wild-type ones demonstrated 15 times fewer survivors under the same stress conditions (Table 1).
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IMPDH homologue alters root morphology, Al penetration and Al-induced callose accumulation
Transgenic seedlings also showed different root morphology under stress conditions when compared to the wild type. After 48 h Al treatment their primary roots did not grow as long as the wild-type ones; however, many morphologically normal lateral roots with numerous root hairs were formed, a phenomenon not observed in the wild type.
To characterize the root phenotype of transgenic seedlings in more detail, Al penetration into the roots was histologically studied by morin staining. After Al treatment primary roots of wild-type plants were heavily and homogenously stained indicating high level of soluble Al chelatable by morin in all root zones. Contrary to the wild type, transgenic roots showed lower staining intensity and zonal pattern of Al distribution (Fig. 8).
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Al-induced callose accumulation in roots was studied by aniline blue staining. After Al treatment, aniline blue fluorescence in the lateral roots of transgenic plants was significantly lower than in the primary roots of the wild type (Fig. 7) and transgenic plant lines. The primary roots of wild-type plantlets were more stress-damaged than the transgenic ones and not able to form lateral roots under the chosen stress regime. Obviously, the formation of lateral roots with a diminished amount of soluble Al penetration and decreased stress-elicited callose accumulation may result in substantial benefit in plant nutrition and growth under stress conditions, since transgenic plants survive the stress regime.
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Discussion |
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Molecular mechanisms of plant tolerance to Al stress have been intensively studied recently. Despite the identification of many genes up-regulated by Al stress, most of them are typical for a general stress response and are not specifically involved in the acquisition of Al tolerance (Ezaki et al., 2000). These investigations were directed to the specific response of soybean plants against Al stress on the level of gene expression. A range of Al chloride concentrations was applied to evaluate the lowest Al concentration causing the first stress responses, but not so toxic that extensive expression of general stress genes could be induced. The Al concentration causing the first visible morphological alterations, such as swelling and damage of the root meristem (data not shown), was 300 µM AlCl3. Higher concentrations caused heavy damage of the root meristem and the root itself. Therefore 300 µM AlCl3 was taken for later Al treatments of soybean roots.
Transcript levels of potential tolerance genes: role of PEPC in soybean resistance to Al
From the eight genes studied that are potentially involved in the mechanisms of plant resistance to Al toxicity, seven did not show any substantial expression difference on mRNA level between stress and non-stress conditions in the tolerant as well as in the sensitive soybean cultivar. Neither a Ca-binding protein, calreticulin, probably involved in the cellular Ca-homeostasis (Kochian, 1995) or in callose accumulation at the plasmodesmata interrupting symplastic Al-transport (Sivaguru et al., 2000), nor Al-induced genes encoding the antioxidation enzyme glutathione-S-transferase (Ezaki et al., 1996), the -subunit of a heterotrimeric G-protein (Ezaki et al., 1996), and different metallothioneins showed an enhanced transcript level upon Al-stress. Therefore they are either not involved in Al tolerance of soybean or they are not stress-regulated on an mRNA level.
The genes encoding enzymes of the tricarboxylic acid cycle, MDH and PEPC, showed different expression activities under stress conditions. The expression of MDH was not increased in Al-stressed roots. PEPC, on the contrary, was up-regulated in root tips during Al stress (Fig. 1). Higher transcript abundance of PEPC in Al-stressed tolerant plants may indicate a functional activity in plant resistance by intracellular production of organic acids. PEPC as a key enzyme of organic acid metabolism in plant cells catalyses conversion of phosphoenolpyruvate to oxaloacetate, which is afterwards metabolized to malate or citrate (Lopez-Bucio et al., 2000). A positive relationship between Al tolerance and organic acid efflux has been reported for several plant species including soybean (Yang et al., 2000; Silva et al., 2001). Exudation of such acids as malic or citric seems to be one of widespread mechanisms of resistance against phytotoxic Al (Delhaize et al., 1993; Ma et al., 1997, 1998; Pellet et al., 1996; Larsen et al., 1998; Ma and Miyasaka, 1998). Since PEPC may be a limiting factor in the metabolite flow towards malic and citric acid production, an increase in its level may be responsible for enhancing organic acid biosynthesis, internal concentration and efflux from roots. On the other hand, the high rates of organic acid efflux from roots are not necessarily related to enzyme activities associated with organic acid metabolism in the TCA cycle (Hocking, 2001), and internal malate or citrate concentrations and their rates of exudation along the roots are not always correlated (Silva et al., 2001; Delhaize et al., 2001). In many cases carbon substrates and enzyme activities do not limit intracellular organic acid production and exudation. Genes encoding the proteins involved in organic acid transport from the roots to the rhizosphere are probably one of the major targets in Al resistance.
Current studies indicate that the exudation of organic anions from the roots is accompanied by the extrusion of protons, presumably via the H+-ATPase pump (Jones, 1998; Neumann et al., 1999, 2000). As in guard cells, PEPC may also be implicated in the generation of H+ via malate production in root tip cells. Under conditions of iron deficiency, an increase of PEPC activity was also connected with H+ extrusion linked to the activity of plasmalemma H+-ATPase and the synthesis of organic acids such as malate and citrate (De Nisi and Zocchi, 2000). Therefore, PEPC functional activity in Al protection could include the regulation of cytosolic pH, the maintenance of amino acid biosynthesis or some influence on H+-ATPase function which together affected plasma membrane charge and function of membrane channels under Al stress (Kochian, 1995).
Al-induced novel stress genes
Comparison of Al-induced gene activities between non-stressed and stressed tolerant and sensitive soybean cultivars was used as a basic selection principle for the genes involved in the Al response. It was considered that if some genes were involved in the general stress response, their expression would be enhanced in both Al-stressed tolerant and sensitive cultivars. For genes specifically involved in protection against toxic Al, the highest expression level was expected in tolerant cultivars under stress conditions, as compared with the stress-treated sensitive ones and non-treated tolerant and sensitive controls. Using this selection principle, two novel genes homologous to TCTP and IMPDH were found to be preferentially up-regulated under stress conditions in the root tips of tolerant soybean cultivars.
In general, TCTP seems to be a cytoplasmic calcium-binding protein. TCTPs or homologous proteins have been found in a wide range of different organisms including human, mouse, rabbit, chicken, earthworm, yeast, and plants. Unlike humans and mice, expression of TCTP-like proteins in plants is probably regulated at the transcriptional level (Sage-Ono et al., 1998). In the earthworm Lumbricus rubellus TCTP is up-regulated under conditions of heavy metal stress, probably promoting the formation of a metal-histidine complex serving as a histamine-releasing factor (Stürzenbaum et al., 1998). However, neither such a mechanism in plants, nor the ability of Al to form histidine complexes like copper are known. Although the functional activities of TCTPs in plants are not understood (Sage-Ono et al., 1998), their remarkable identities on the protein level indicate high conservation suggesting essential functions. Suggestion of a possible function of the soybean TCTP-like protein encoded by the cDNA clone 58 is difficult. It is possible that this protein is involved in the maintenance of Ca homeostasis in stressed plant cells, because the essential Ca2+ concentration in different cellular compartments plays an important role in the amelioration of Al-caused damage (Kochian, 1995).
The protein encoded by cDNA 633 is homologous (average identity 67%) to the C-terminal part of inosine-5'-monophosphate dehydrogenases (IMPDH) of plants and animals. IMPDHs catalyse the rate-limiting step in the de novo biosynthesis of guanine nucleotides (Huberman et al., 1995). They play an essential role in providing necessary DNA and RNA precursors and are also involved in signal transduction pathways mediating cell differentiation in humans and animals (Jenkins et al., 1993). Furthermore, it has been suggested that inosine-5'-monophosphate oxidation by IMPDH might be the predominant pathway leading to xanthine and the formation of ureides in plants (Atkins et al., 1985). The putative protein might be involved in the enhancement of nucleic acid biosynthesis, as known IMPDHs, or may take part in GTP exchange as a part of signal transduction pathways (Jayaram et al., 1999) in stressed plant cells. The C-terminal topology of the myristoylation sites indicates a possible membrane association of the protein (Thomson and Okuyama, 2000). However, the protein encoded by cDNA 633 is homologous only to the C-terminal part of IMPDHs and, therefore, it is not clear whether its function is identical or similar to IMPDHs.
IMPDH homologue mediates Al stress resistance in transgenic plants
Several genetic engineering experiments have been performed to acquire Al tolerance in plants by over-expression of foreign genes probably involved in plant resistance. Expression of a Pseudomonas aeruginosa gene coding for citrate synthase revealed enhanced Al resistance and benefit of phosphorus uptake in transgenic tobacco plants (de la Fuente et al., 1997; Lopez-Bucio et al., 2000). Later, characterization of these transgenic plants (Delhaize et al., 2001) demonstrated, however, that cytoplasmic expression of the bacterial citrate synthase gene is unlikely to be a robust and reproducible way to mediate Al tolerance. Several Al-induced genes transformed into Arabidopsis plants conferred a degree of Al-resistance (Ezaki et al., 2001). Compared with the wild type, transgenic plants showed increased root growth, diminished Al-induced oxidative damage in root cells and decreased stress-elicited callose accumulation under relatively low Al concentrations. It was suggested that plants possessing a higher degree of resistance can be created by a combination of different Al-induced genes (Ezaki et al., 2001). Until now the expression of Al up-regulated genes in plants did not lead to dramatically enhanced Al tolerance of transgenic plants or contribute to the functional analysis of the genes specifically involved in the tolerance mechanisms.
To check the functional role of the IMPDH homologue in stress response, cDNA 633 was expressed in Arabidopsis cv. Columbia plants, which resulted in about 15-fold increased resistance of transgenic plants against Al (Table 1). After 48 h AlCl3 treatment, transgenic plants were able to form numerous lateral roots, which probably cause substantial benefit in maintaining water, nutrition uptake and surviving the stress.
After stress treatment, seedlings of different transgenic lines showed similar patterns of Al distribution in the root tips, which significantly differed from that of the wild type (Fig. 8). Compared with the wild type, the content of soluble Al was significantly lower in the root tips of transgenic plants. A lower cytoplasmic Al content in this zone is probably connected with the enhanced Al resistance of transgenics. Lower soluble Al content was also observed in the other root regions, but the difference was not as high as for the division and elongation zone. A dramatic decrease of Al content in the division and elongation zone of transgenic roots seems to be quite important because it is the primary target of Al toxicity (Ryan et al., 1992, 1995).
Diminished Al-elicited callose formation in the lateral roots of transgenic as compared to the primary roots of wild-type plants without lateral root formation also suggests an enhanced ability of the lateral roots to overcome Al stress. Substantially lower callose biosynthesis in lateral roots of transgenic plants corresponds to lower Al penetration into the division and elongation zone of transgenic primary roots after shorter stress treatment (Fig. 8), whereas extensive callose formation in primary roots of the wild type correlated with a high amount of intracellular soluble Al inside the roots. Changes of root morphology, lowered Al penetration and enhanced stress resistance of lateral roots are responsible for survival under Al-stress conditions by maintenance of metabolic activities in root cells as well as water and nutrient uptake.
Whether the IMPDH homologue is involved in Al resistance mechanisms, is unclear. Lower free Al content in the cell division and elongation zone of transgenic roots should significantly diminish Al toxicity and enable the formation of a complex root system saving nutrition and water uptake. It is not quite certain whether the IMPDH homologue belongs to the apoplastic or symplastic mechanism of resistance. Further characterization of Al-induced genes such as PEPC, TCTP or IMPDH together with their influence on Al-tolerance has to be performed in the future to get more insight into the molecular mechanisms of plant resistance against the phytotoxic Al ion.
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Acknowledgements |
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The authors would like to thank Dr Annegret Tewes (IPK, Gatersleben) for providing the cell suspension culture material of Al-stressed tolerant soybean cv. Wilis. We also would like to thank S Miroshnichenko (IPK, Gatersleben) for providing pRTRA3/7 plasmid DNA. This work was supported by TÜV Rheinland/BMBF Project 0319179E.
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