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a Institute of Plant Physiology, Martin-Luther Univ. Halle-Wittenberg, Weinbergweg 10, 06120 Halle, Germany
b Institute of Biochemistry, Martin-Luther-Univ. Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle, Germany
* Corresponding author (klaus.humbeck{at}pflanzenphys.uni-halle.de).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: aa, amino acids • bp, base pairs • EST, expressed sequence tag • MS, Murashige–Skoog • MT, metallothionein • PS, photosystem • RT-PCR • reverse transcription polymerase chain reaction.
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Therefore, metal uptake, transport, insertion into, and liberation from their protein complexes must be carefully regulated. In leaves, efficient mechanisms for maintaining metal homeostasis must function, especially during early phases of leaf development when metals are inserted into the metalloproteins and during leaf senescence when these metals are released again. The most prominent process during leaf senescence is the breakdown of the chloroplasts, when metals like Fe and Cu are liberated from their apoproteins and transferred from senescing leaves into other parts of the plant (Mauk and Noodén, 1992; Drossopoulos et al., 1994, 1996; Hocking, 1994).
Not only metal transfer during plant development but also environmental pollution and the natural occurrence of heavy metals require efficient mechanisms of metal detoxification. Therefore, plants have developed a broad variety of mechanisms for metal homeostasis and metal detoxification both for metals like Zn, Fe, and Cu, which are essential in trace amounts but toxic in only slightly quantities, and for nonessential metals such as Pb, Al, and Cd, which are not required for plant cellular functions.
Among the different metal binding factors, metallothioneins (MTs) play a major part in the processes of plant metal homeostasis and detoxification (Cobbett and Goldsbrough, 2000). These peptides are cysteine rich and low in aromatic amino acids with molecular weights between 6 and 8 kDa, and they show typical clusters of cysteines in the amino- and carboxy-terminal parts of the protein. On the basis of their characteristic arrangement of cysteines, plant metallothioneins can be divided into four different types: MT 1, MT 2, MT 3, and MT 4 (Robinson et al., 1993; Cobbett and Goldsbrough, 2002).
The different MTs vary dramatically in their gene expression. In plants, some MTs are induced by various elicitors like pathogens or heavy metals, others are differentially expressed in different tissues and at different developmental stages (for detailed review, see Cobbett and Goldsbrough, 2002). Besides MT gene expression studies on rice (Oryza sativa L.) leaves (Hsieh et al.,1995, 1996; Yu et al., 1998; Matsumura et al., 1999; Wong et al., 2004; Zhou et al., 2005), no comprehensive studies on MT gene expression in leaves of other crop plants like wheat (Triticum aestivum L.), maize (Zea mays L.), or barley (Hordeum vulgare L.) have been reported. In this study, we analyzed the MT gene expression in barley leaves during natural leaf development, under metal-deficiency conditions, and after exposure to excess concentrations of heavy metals.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For senescence analysis, plants were grown in soil (ED73, Patzer, Sinntal-Jossa, Germany) containing 4 g L–1 Chrysal fertilizer (Multicote, Paka & Chrysal, Naarden, the Netherlands) in 6-L Mitscherlich pots (Stoma, Siegburg, Germany) under the same environmental conditions as described by Miersch et al. (2000). Primary leaves were harvested 9, 21, and 39 d after sowing, immediately frozen in liquid N2 and stored at –80°C.
Photosystem II Efficiency
Chlorophyll fluorescence measurements after dark adaptation for 20 min were performed as described by Humbeck et al. (1996) using a chlorophyll fluorometer (Mini PAM, Walz, Effeltrich, Germany). The measurements were made on the middle region of intact leaves 8 h into the light period. Mean values of the ratio of variable fluorescence/maximal fluorescence (Fv/Fm) are always based on measurements with 10 different plants from at least two independent growth experiments.
Chlorophyll Content
Relative chlorophyll content per unit leaf area was determined using a SPAD analyzer (Soil Plant Analysis Development, Minolta by Hydro Agri, Dülmen, Germany) which measures transmission of wavelengths absorbed by chlorophylls in intact leaves (Wood et al., 1993). Each data point represents the mean value of 10 measurements with different plants from at least two independent experiments.
Reverse Transcription-Polymerase Chain Reaction
Reverse transcription-polymerase chain reaction (RT-PCR) was performed with a one-step RT-PCR kit according to the manufacturer's recommendation (One-Step RT-PCR, Qiagen, Valencia, CA) with 1 µg of total RNA extracted from leaves at either different developmental stages or after exposure to heavy metals, as shown in Table 1. The following oligonucleotides were used as primers:
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for 5'-ATGTCTTGCARYTGTGAATCAAGC-3' and rev 5'-GCAGTTGCAAGGTCGCACTT-3'
HvMT-2a:
for 5'-ATGTCTTGCTGCGGAGAAAACTGC-3' and rev 5'-GCGGCAGCGCCTGCAAGT-3'
HvMT-2b:
for 5'-ATGTCGTGCTGCGGAGGAARCTGC-3' and rev 5'-GCAGMTGCAYGGGTTGCAG-GTGC-3'
HvMT-3a:
for 5'-AGGCACTCTCCGATCACAAG-3' and rev 5'-ACGGCAACAACACAACAGAC-3'
HvMT-4a:
for 5'-AACGCCTGAAAAGATCGAGA-3' and rev 5'-ACTTTCCACACACGCACAAA-3'
Obtained RT-PCR products were sequenced using the BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit and the ABI Prism 370 DNA-Sequenzer (both Applied Biosystems, Foster City, CA).
RNA Isolation and Northern Analysis
Total RNA of whole leaves was isolated according to the method of Chirgwin et al. (1979). The RNA content was quantified spectrophotometrically and 15 µg of each sample electrophoretically fractioned on 1% (w/v) agarose gels containing formaldehyde. The RNA was transferred by pressure blotting (Stratagene, Amsterdam) onto positively charged nylon membranes (Roti-plus, Roth, Karlsruhe, Germany).
Membranes were prehybridizied at 50°C for 1.5 h in a solution consisting of 50% (v/v) deionized formamide, 74.7 mM sodium citrate at pH 7.0, 74.7 mM NaCl, 50 mM sodium phosphate, 2% (w/v) N-laurylsarcosine, and 7% (w/v) sodium dodecylsulfate. After discarding the prehybridization solution, hybridization was performed at 50°C overnight in a solution consisting of the same ingredients as the prehybridization solution plus the digoxygenine (DIG)-labeled probe. All gene expression data were confirmed by at least two independent experiments.
The DIG-labeled probes were prepared by incorporating DIG-labeled deoxyuridine triphosphate (dUTP; Boehringer, Mannheim, Germany) in PCR reactions using the above-mentioned oligonucleotide sequences and obtained RT-PCR products. The DIG-labeled bands were detected by chemiluminescence using chemiluminescence substrate phenyl-phosphate disodium (Boehringer, Mannheim, Germany).
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The HvMT-1a gne (AJ51344, 225 base pairs [bp]) was isolated from primary leaves of Cd-treated plants. The derived protein sequence of 75 amino acids (aa) shows 99% (74 of 75 aa) homology to a type 1 MT from wheat (wali1, P43400) that is induced in roots and leaves under Al stress (Snowden and Gardner, 1993). The RT-PCR experiments with RNA from leaves using primers designed for the already known barley MT ids-1 (in this study renamed to HvMT-1b), which is induced by Fe deficiency in roots (Okumura et al., 1991), did not result in any amplification of the HvMT-1b cDNA. This mRNA could only be detected in roots and was therefore not further investigated. Different from HvMT-1a, the cDNA of HvMT-1d (AJ555615, 222 bp) was isolated not from leaves but from roots of Cd-exposed young barley seedlings. A database search revealed that this putative MT type 1 shows 99% identity to the barley EST BQ766316 (73 of 74 aa).
By the RT-PCR approach, two barley type 2 MTs could also be identified. The HvMT-2a MT (AJ511345, 234 bp), which was isolated from 39-d-old barley leaves, represents a putative type 2 MT homolog with a predicted amino acid sequence of 78 aa. Scanned against the databases, HvMT-2a shows 72.5% (58 of 80 aa) identity to rice OsMT2c. The OsMT2c MT is constitutively expressed in the stem of rice plants (Yu et al., 1998). The 244 bp coding sequence of HvMT-2b (AJ511346), also isolated from old leaves of barley, encodes a putative type 2 MT homolog with a predicted amino acid sequence of 81 aa, which shows 75% identity (60 of 80 aa) to Sandberg bluegrass (Poa secunda J. Presl) MT 2 (AAK38824), which is known to be expressed during illumination of excised leaves (Wei et al., 2002).
The HvMT-3a gene (AJ555613, 186 bp) was isolated from young primary barley leaves. It encodes a putative MT protein sequence of 62 amino acids and shows 67% (43 of 62 aa) identity to OsMT3a (AC135920). The 231 bp coding sequence of HvMT-4a (AJ555614) obtained by RT-PCR with RNA from old leaves represents a putative MT type 4 homolog with a predicted sequence of 77 amino acids. The derived amino acid sequence of HvMT-4a shows 89% identity (72 of 81 aa) to wheat Ec (CAA48350), induced during embryogenesis (Kawashima et al., 1992). The Ec gene was the first MT identified in plants (Hanley-Bowdin and Lane, 1983).
Following the nomenclature proposed by Cobbett and Goldsbrough (2002), all known barley MTs, including the four newly identified MTs in this study, can be classified into two type 1 (HvMT-1a, HvMT-1d), two type 2 (HvMT-2a, HvMT-2b), one type 3 (HvMT-3a), and one type 4 MT (HvMT-4a). The results show that together with the already-known barley MTs HvMT-2c (B22EL8), HvMT-1b (ids-1), and HvMT-1c (EST BQ768005), at least nine MTs are present in this Poaceae species.
This number of barley MTs equals the number of MTs reported in rice by Wong et al. (2004), who renamed the genes according to the nomenclature in a more structured way. In this study, we also propose to change the existing names of the two known barley MTs according to the Cobbett and Goldsbrough (2002) MT nomenclature. Table 2 summarizes the different barley and rice MTs and their proposed novel gene names.
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As shown in Fig. 4, the different barley MTs exhibited quite different gene expression patterns. The HvMT-1a gene was strongly induced during leaf senescence and also by exposure of the seedlings to toxic concentrations of Cu and Cd, regardless of whether the plants were grown in MS medium or under putative metal-deficient conditions in tap water. The HvMT-2a gene, however, does show constitutive gene expression. This mRNA was detected by Northern analysis and RT-PCR in all leaf samples in high abundance.
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The HvMT-3a gene showed no difference in gene expression in leaves of different developmental stages (9, 21, and 39 d, plants grown in soil) and during heavy metal stress when seedlings were grown in MS nutrient medium (Fig. 4). High levels of HvMT-3a mRNA however, could be detected in the control plants of the heavy metal stress experiment and in plants treated with Cd when seedlings were grown in tap water (Fig. 4, right column). In this experiment, the control, the Cu- and the Cd-treated plants were grown hydroponically for 10 d in tap water without any nutrient supply. Therefore, these three samples were putatively metal deficient. Since upregulation of HvMT-3a was not detected in samples exposed to Cu, a second experiment was performed to test the hypothesis that expression of HvMT-3a is linked to Cu deficiency (Fig. 5). The HvMT-3a gene was strongly induced in leaves of barley plants that were grown in tap water for 12 d without addition of Cu to the medium (Fig. 5, Lane 1). In primary leaves of plants that were grown for 10 d in water and then exposed to Cu for 48 h, HvMT-3a expression was clearly downregulated. Inhibition of HvMT-3a expression already occurred at a 2000-fold lower Cu concentration of 0.5 µM (Fig. 5, Lane 2) and was also inhibited by toxic concentrations of 1 mM Cu plus 1 mM Cd (Fig. 5, Lane 5).
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Different from known experiments where Poaceae seedlings were exposed to heavy metals during development for 96 h or longer (wheat, Snowden and Gardner, 1993; barley, Brune and Dietz, 1995; Blinda et al., 1997), in this study barley seedlings were exposed to high toxic concentrations of heavy metals for a short period of 48 h to avoid the interference of natural leaf senescence with the response to heavy metal treatment.
For the physiological characterization, the two photosynthesis parameters, chlorophyll content and PS II efficiency, displayed rapid changes after exposure of plants to heavy metals (this study), and sensitively indicated the course of leaf senescence (Miersch et al., 2000).
The continuous loss of chlorophyll of the barley primary leaf starting 10 d after seed germination reveals natural leaf senescence. The senescence-like process of chlorophyll degradation occurred also by treating barley plants with the toxic concentration of 1 mM Cd or Cu. Here, a 30% loss of chlorophyll, which during natural primary leaf senescence takes about 3 wk, can be observed within 48 h. The maintained efficiency of remaining PS II centers during both leaf senescence and short-term heavy metal treatment indicates, however, the maintenance of cellular integrity and, therefore, the ability of the plant to cope with these circumstances. Interestingly, the primary leaves of seedlings grown under metal deficiency in tap water showed lower total chlorophyll content than the MS plants, but they did not show a more severe stress response during heavy metal treatment.
To identify putative factors of metal homeostasis and detoxification in leaves, we looked for genes coding for MTs expressed during leaf senescence and heavy-metal treatment. Our results show that at least nine different genes for MTs are present in barley, and that they group, according to the MT nomenclature by Cobbett and Goldsbrough (2002), into four type 1 (HvMT-1a, HvMT-1b, HvMT-1c, and HvMT-1d), three type 2 (HvMT-2a, HvMT-2b, and HvMT-2c), one type 3 (HvMT-3a), and one type 4 (HvMT-4a) MT.
Like its MT type 1 homologs in wheat (wali-1, Snowden and Gardner, 1993) and rice (OsMT-1a, Hsieh et al., 1995), the barley HvMT-1a is expressed during heavy-metal stress and, like OsMT-1a, also during leaf senescence (Hsieh et al., 1995). These MTs might play a role in maintaining metal homeostasis, since their gene expression is induced either by metal uptake or by liberation of metals during senescence-dependent degradation of the plastids. Interestingly, HvMT-1a does not show enhanced gene expression under metal-deprivation conditions, other than its isoform HvMT-1b, which is expressed during Fe deficiency in barley roots (Okumura et al., 1991). In our experiments, neither HvMT-1b nor the type 1 MT HvMT-1d could be amplified by RT-PCR with mRNA extracted from leaves. The latter type 1 MT was detected in roots after Cd treatment of barley seedlings (data not shown).
In leaves, the newly identified type 3 HvMT-3a gene induction clearly depends on metal deficiency. The HvMT-3a gene expression results from Cu deficiency, however, not from Fe deficiency as HvMT-1b does (Okumura et al., 1991). For both conditions, the lack of essential metals may cause expression of genes encoding proteins involved in either metal uptake or transport. This response to metal deficiency would result in better uptake and would therefore be an adaptation mechanism. The Poaceae equivalent for HvMT-3a, the two rice MTs OsMT3a and OsMT3b and the wheat EST (CA687517), have so far not been analyzed with respect to their expression patterns.
The regulation of MT 2 genes in leaves differs from MT 1 genes. While transcripts of the Poaceae MT 1 genes are not highly abundant in non-metal-exposed or nonsenescent tissue (rice OsMT-1a, Hsieh et al., 1995; wheat wali1, Snowden and Gardner, 1993; barley HvMT-1a; red fescue [Festuca rubra L. subsp. rubra] mcMT, Ma et al., 2003), the mRNA levels of type 2 MTs show constitutive expression in non-metal-exposed leaves and are either constitutive or downregulated by stress (rice, OsMT-2a, OsMT-2b, Hsieh et al., 1996; Wong et al., 2004; barley, HvMT-2a, HvMT-2b, this study). The high abundance of HvMT-2a mRNA in barley leaves of different developmental stages matches the results in etiolated rice seedlings obtained by serial analysis of gene expression, where the rice MT-2 homolog OsMT-2b was the most abundant transcript (Matsumura et al., 1999). Like HvMT-2c (Klemsdal et al., 1991; White et al., 2006), the maize MT type 2 (Charbonnel-Campa et al., 2000) is expressed during embryogenesis, but its expression during senescence and during exposure to metals has not been investigated so far.
The wheat Ec (Hanley-Bowdin and Lane, 1983; Kawashima et al., 1992) and the rice OsMT-II-a (Zhou et al., 2005), which are now classified as type 4 MTs, are expressed in developing seeds. As in rice (Zhou et al., 2005), the barley gene HvMT-4a showed no gene expression in untreated or metal-treated young and mature leaves. The HvMT-4a gene was isolated by RT-PCR with RNA from senescent leaves, however, whereas the same PCR with RNA from 4-d-old seedlings did not show any PCR product (data not shown). This indicates that HvMT-4a may somehow be involved in the senescence process, but its transcript level is very low. Furthermore, wheat Ec and rice OsMT4 gene expression can be triggered by exogenous application of abscisic acid (ABA) (Kawashima et al., 1992; Zhou et al., 2005). In leaves, ABA accumulates greatly during osmotic stress, which occurs during late stages of leaf senescence. Whether HvMT-4a plays a role in leaves during special phases of senescence, and whether it is also induced in response to ABA and during embryogenesis, have to be clarified in future experiments.
Our results show that during leaf development, metal deficiency, or heavy-metal treatment, some MT genes (HvMT-1a, -2b, and -3a) are differentially expressed in barley leaves. Other barley MTs (HvMT-1b, -1c, -1d, and -2c) are expressed in other tissues like the roots or during other stages of plant development, indicating specific functions of the different MTs during development and stress response.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication July 27, 2006.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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