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Physiology and Biochemistry |
2Department of Earth, Ecological, and Environmental Sciences (MS 604), University of Toledo, Toledo, Ohio 43606 USA; 3Department of Biology, Syracuse University, Syracuse, New York 13244 USA
Received for publication November 14, 2003. Accepted for publication April 23, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: heat-shock proteins • heavy metals • photosynthesis • stress proteins
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Salt et al., 1998
; Taiz and Zeiger, 1998
). Plants exposed to high levels of heavy metals typically accumulate the metals in their tissues (both roots and shoots), often to toxic levels that decrease growth or reproduction. The mechanisms utilized by plants to protect cells from excess heavy metals are not completely understood, but are known to include internal and external chelators, efflux pumping, vacuolar compartmentalization, and heat-shock proteins (Salt et al., 1998
; Cobbett, 2000
; Hall, 2002
).
Photosynthesis is typically decreased by elevated levels of heavy metals, but the specific effects of a given heavy metal on photosynthesis vary among species, preventing broad generalizations about metal effects on photosynthesis. For example, copper has been shown to negatively affect components of both the light reactions (e.g., photosystem II [PSII], thylakoid membrane structure, chlorophyll content) (Gupta and Singhal, 1995
; Ralph and Burchett, 1998
; Szalontai et al., 1999
; Pätsikkä et al., 2002
; Vinit-Dunand et al., 2002
) and CO2-fixation reactions (Stiborova et al., 1986
; Lidon and Henriques, 1991
; Angelov et al., 1993
; Moustakas et al., 1994
). However, in studies that examined both light and CO2-fixation components, the relative sensitivity of each to Cu varies among studies (Stiborova et al., 1986
; Lidon and Henriques, 1991
; Angelov et al., 1993
; Moustakas et al., 1994
). Similar observations have been made for other metals, such as lead (Stiborova et al., 1986
; Moustakas et al., 1994
; Singh et al., 1997
; Ralph and Burchett, 1998
; Szalontai et al., 1999
).
As mentioned, a general response of plants to elevated levels of heavy metals appears to be increased synthesis of various heat-shock proteins (Hsps) (e.g., Tseng et al., 1993
; Neumann et al., 1994
, 1995
; Barque et al., 1996
; Wollgiehn and Neumann, 1999
; Hall, 2002
). However, the specific cell components or processes targeted by Hsps during metal stress are not yet identified. Both cell membranes and proteins are primary sites of damage to cells during heavy metal stress, and limited evidence suggests that Hsps function to protect cell membranes from metal damage (Hall, 2002
). In addition, given the well-characterized roles of Hsps in the protection and repair of soluble proteins during heat stress (e.g., Parsell and Lindquist, 1994
) Hsps may function in this way during metal stress too. Recently, the chloroplast small (low molecular mass) Hsp has been shown to protect photosynthesis during heat, oxidative, and photoinhibitory stress, by protecting PSII or other aspects of thylakoids (Heckathorn et al., 1998
; Lee et al., 1998
, 2000a
, b
; Miyao-Tokutomi et al., 1998
; Downs et al., 1999a
, b
; Nakamoto et al., 2000
; Török et al., 2001
). Also, chloroplast smHsps probably protect photosynthesis via more than one mechanism: by preventing irreversible protein aggregation (Török et al., 2001
), by stabilizing chloroplast membranes (Török et al., 2001
), and possibly as site-specific antioxidants (Hamilton and Heckathorn, 2001
). Given this, and that heavy metals damage both membrane and soluble phases of chloroplasts through multiple mechanisms that include protein denaturation and oxidative damage (Hall, 2002
), we hypothesized that the chloroplast smHsps are produced when heavy metals accumulate in the chloroplast and damage photosynthesis and that smHsps help protect photosynthesis from excess heavy metals. To test this, we exposed Zea mays plants to Cu, Ni, Pb, and Zn at levels sufficient to decrease in vivo net photosynthesis (Phn) (and chlorophyll content and PSII function, too), then determined if chloroplast smHsp levels were elevated with metal exposure. Having positive evidence of metal-induced decreases in Phn and increases in chloroplast smHsps, we directly determined if chloroplast smHsps could protect photosynthetic electron transport (Phet) when added to thylakoids exposed to high levels of heavy metals. In vivo protection of Phn by smHsp was confirmed in two, previously characterized, near-isogenic genotypes of Agrostis stolonifera differing in the number of chloroplast smHsp genes expressed and in the amount of smHsp protein produced during stress (Park et al., 1996
; Luthe et al., 2000
; Heckathorn et al., 2002
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A heat-sensitive progenitor of Agrostis stolonifera Huds. var. palustris (creeping bentgrass) and a near-isogenic heat-tolerant selection mutant were propagated vegetatively. These two previously characterized genotypes differ in the number of chloroplast smHsp genes expressed (one and two, respectively). Consequently, the tolerant genotype produces approximately twice as much chloroplast smHsp protein as the sensitive genotype (Park et al., 1996
; Luthe et al., 2000
; Heckathorn et al., 2002
). Increased smHsp accumulation in the tolerant genotype is genetically linked to increased plant thermotolerance (Park et al., 1996
) and results in the increased tolerance of Phet and PSII to heat stress (Heckathorn et al., 2002
). Vegetative plants were grown at 22°C days and 18°C nights for several weeks in pots as described, but at ca. 200 µmol · m–2 · s–1 PAR. Individual adult plants were then transferred to aerated flasks containing 2000 µmol/L Ni or Pb and then monitored after 4 d.
Protein extraction and content
Proteins were extracted from tissues or chloroplasts in an extraction buffer containing 1% sodium dodecyl sulfate (SDS), 100 mmol/L Tris pH 8.0, 10 mmol/L EDTA, 1 mmol/L 
-amino caproic acid, 1 mmol/L phenylmethylsulfonylfluoride (PMSF), 1 mmol/L benzamidine, 10 mmol/L ascorbate, 10 mmol/L dithiothreitol, 1 µmol/L antipain and leupeptin, 10% glycerol (v/v), 10% sucrose (m/v), 2% polyvinylpolypyrrolidone (PVPP) and 2% polyvinylpyrrolidone (PVP) (m/v), and 0.05% bromophenol blue. Leaf proteins were extracted by grinding tissue (0.1 g) in liquid N2, then in extraction buffer (1 mL) with a mortar and pestle. Chloroplast proteins were then extracted by solubilizing isolated chloroplasts in extraction buffer. Protein extractions were heated for 2 min at 75°–85°C and then centrifuged at 21 000 x g for 2 min to remove insoluble debris. Protein concentration of each sample was determined in triplicate by the Coomassie dye-binding method of Ghosh et al. (1988)
, using bovine serum albumin as a standard and using a desktop scanner and National Institutes of Health imaging software (Scion) to perform densitometry.
Electrophoresis and immunoblotting
Protein extracts were fractionated by SDS-PAGE, using 16 x 20 x 0.15-cm 15% gels; equal total protein was loaded per lane (40 µg). Following SDS-PAGE, the proteins were transferred to nitrocellulose membranes by electroblotting for 90 min at 350 mA. The membranes were then probed with protein-specific antibodies and secondary antibodies conjugated to alkaline phosphatase. Antibodies to the chloroplast small Hsps were polyclonal rabbit antibodies raised against oligopeptides of conserved sequences (described in Heckathorn et al., 2002
). Secondary antibodies were detected with a colorimetric system consisting of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (densitometry performed as above).
Chloroplast isolation
Chloroplasts were isolated by a combination of differential and density gradient centrifugation, using a modification of the method described previously (Heckathorn et al., 1998
). As before, leaves were ground, and chloroplasts were partially purified by differential centrifugation (5000 x g for 10 min), in a buffer containing 350 mmol/L sorbitol, 50 mmol/ L Hepes (pH 7.75), 5 mmol/L EDTA, 5 mmol/L ascorbate, 2 mmol/L dithiothreitol, and 2% PVP (m/v). The resulting crude chloroplast pellet was resuspended in this same buffer, and the chloroplasts were subjected to density gradient centrifugation (7000 x g for 10 min) using a step-gradient containing 0.5, 1.0, 1.5, and 2.0 mol/L sucrose in 50 mmol/L Hepes (pH 7.75). Intact and broken chloroplasts were collected separately from the gradients with a pipet, repelleted by low-speed centrifugation (5000 x g for 10 min), resuspended in a small volume (10 : 1 volume of buffer : sample) of 1.0 mol/L sucrose in 50 mmol/L Hepes (pH 7.75), and then stored at –70°C. Chloroplast or thylakoid samples analyzed by SDS-PAGE were thawed and resuspended in protein extraction buffer (as earlier), while samples used in electron transport assays were diluted to 100 mmol/L sucrose and 25 µg chlorophyll/mL as described later.
Photosynthetic measurements
Photosystem II function of intact leaves was monitored by determining the ratio of variable-to-maximum fluorescence (Fv/Fm) of dark-adapted leaves with a pulse-amplitude-modulated fluorometer (model PAM 101/103; Walz, Effeltrich, Germany). Net photosynthesis of intact leaves was monitored by measuring net CO2 exchange with an infrared gas analyzer-based photosynthesis system (model 6200; LI-COR, Lincoln, Nebraska, USA) (e.g., Heckathorn et al., 1996
, 2002
). Net photosynthesis and Fv/Fm were measured in the growth chamber at midday (1300–1500 h) on recently fully expanded leaves; net photosynthesis was measured at 400 or 200 µmol · m–2 · s–1 PAR (for corn and bentgrass, respectively) and 425 ± 20 µmol/mol CO2, while Fv/Fm was measured after 30 min of dark adaptation.
Whole-chain photosynthetic electron transport was measured in isolated thylakoids by monitoring O2 evolution from PSII in the presence of potassium ferricyanide (K3Fe(CN)6), an artificial electron acceptor that receives electrons from PSI (thus intercepting flow from PSI to NADP+; Allen and Holmes, 1986
). O2 evolution was monitored using a Clark-type O2 electrode (Hansatech, Norfolk, England). Isolated thylakoids were resuspended to 25 µg chlorophyll/mL in 50 mmol/L HEPES (pH 7.75), 100 mmol/L sucrose, 4 mmol/ L MgCl2, 4 mmol/L NaCl, 0.01% Triton X-100, and 4 mmol/L K3Fe(CN)6 (added just prior to measurement) (Allen and Holmes, 1986
). Electron transport was monitored for 15 min at 25°C, at either 150 or 1300 µmol · m–2 · s–1 PAR, with or without Pb or Ni (either 500 or 1000 µmol/L), and with or without purified chloroplast smHsp (in 50 µL aliquot) (as in Heckathorn et al., 1998
; Downs et al., 1999b
); rabbit whole serum was used as a negative protein addition control (50 µL). We examined only two of the four metals in these in vitro experiments, due to limited amount of purified smHsp, and randomly chose Ni and Pb as representative. Protection of electron transport by purified smHsp was titrated, and maximum protection was achieved with 1.125 mol of smHsp per mol of PSII, a stoichiometry of approximately 1 : 1 (assuming 250 chlorophyll molecules per PSII and PSI each and a PSII/PSI ratio of 1.8 [Buchanan et al., 2000
]; tomato smHsp MW = 20 000 [Waters et al., 1996]
).
Chlorophyll concentration of isolated thylakoids and leaves after extraction in dimethyl sulfoxide (DMSO) was determined spectrophotometrically using the equations of Barnes et al. (1992)
. For leaves, segments from the mid-length region of recently expanded leaves were removed using a cork borer (avoiding the mid vein), then leaf pieces were incubated in DMSO in the dark at 60°C for 1.5 h prior to measurement.
Purification of smHsp
Chloroplast smHsp was purified from heat-stressed tomato plants (Lycopersicon esculentum Mill. cv. Condine Red), using a method modified from Lee and Vierling (1998)
. Plants were heat stressed for 8 h during the day, and then chloroplasts were isolated from leaves as earlier, except that a two-step sucrose gradient was used (0.5 and 2.0 mol/L), resulting in a mixture of broken and intact chloroplasts. These chloroplasts were resuspended in a protein purification buffer (PP buffer) containing 20 mmol/L Tris (pH 8.0), 1% glycerol, 1 mmol/L EDTA, 0.1 mmol/L PMSF, 0.1 mmol/ L benzamidine, 0.1 mmol/L -amino caproic acid, 0.1 µmol/L leupeptin, 0.1 µmol/L antipain, and 0.1% Triton X-100. Chloroplast proteins were then fractionated by ammonium sulfate precipitation [25, 50, 75, and 100% (NH4)2SO4,] and collected by centrifugation at 10 000 x g for 10 min; chloroplast smHsps precipitated at 75% (determined by SDS-PAGE and immunoblotting). Following this, the smHsp-enriched fraction was resuspended in PP buffer and then diluted 100-fold with 20 mmol/L Tris (pH 8.0), to ensure that residual (NH4)2SO4 was less than 30 mmol/L. The proteins in this fraction were separated by anion-exchange chromatography, using DEAE Sepharose (CL-6B; Sigma, St. Louis, Missouri, USA) in a 13 x 2.5-cm gravity flow column. Proteins were eluted from the column using a step-gradient of NaCl in 20 mmol/L Tris (pH 8.0) (100, 200, 300, 400, and 500 mmol/L NaCl; 50 mL per step) and collected in 10-mL fractions. Anion-exchange fractions containing smHsp (five fractions between 400–500 mmol/L NaCl) were determined by SDS-PAGE and immunoblotting. These fractions were pooled, desalted by gel-filtration chromatography (1. 5 x 20-cm gravity flow column with Sephadex G-25; Amersham Biosciences, Uppsala, Sweden) using 10 mmol/L sodium phosphate (pH 7.3) for elution/buffer exchange (protein elution monitored spectrophotometrically). Proteins were then fractionated by hydroxyapatite chromatography (1.5 x 15-cm gravity flow column with Bio-Gel HTP; Bio-Rad, Hercules, California, USA). Proteins were eluted from the column using 50 mL steps of 25, 50, 100, 200, and 400 mmol/L sodium phosphate (pH 7.3). Spectrophotometric analysis of each eluted fraction indicated three protein peaks, and immunoblotting indicated that the last of these three peaks (somewhere between 100 and 200 mmol/L sodium phosphate) contained only the chloroplast small Hsp (confirmed by silver staining). This fraction, containing small Hsp at a protein concentration of 0.0017 g/L, was stored at –70°C and used directly for electron transport assays (final concentration described above).
Statistical analyses
Results were analyzed by analysis of variance (ANOVA), first by including all main effects and their interactions in the models, and then, when appropriate (i.e., when significant main effects were observed at P < 0.05), only single levels of main effects (e.g., days) were included in ANOVA analyses. Differences among levels of main effects were analyzed by Tukey's test or by a t test, following significant ANOVA results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Downs et al., 1999b
; Hamilton and Heckathorn, 2001
). Heavy metal accumulation damages cell components directly (e.g., by poisoning enzymes) and indirectly (e.g., by inducing oxidative damage to proteins and lipids) (Hall, 2002
). Therefore, the production of certain Hsps might be predicted to increase during heavy metal stress in plants or other organisms. However, based on the few studies that have investigated the effects of heavy metals on production of heat-shock proteins in plants, the increased production of Hsps is indeed a general plant response to heavy metal accumulation (reviewed in Hall, 2002
). These studies also indicate that members of the Hsp70 and small Hsp groups are especially responsive to heavy metals, and there is some evidence to suggest that one of the key functions of Hsps during heavy metal stress is to protect the cell membrane (a primary site of heavy metal damage) (Hall, 2002
). However, with few exceptions, the specific Hsps induced by heavy metals and the cellular location of these proteins are unidentified, as are the specific roles or cell targets of Hsps during metal stress (Hall, 2002
).
In the present study, the chloroplast small Hsp, which is typically not produced by plants in the absence of stress, was induced by exposure to all four heavy metals examined (Cu, Ni, Pb, Zn) to levels comparable to those induced by severe acute heat stress (Fig. 2). In corn, purified chloroplast small Hsp was able to protect photosynthetic electron transport in vitro from direct toxic effects of Pb (Fig. 3). In A. stolonifera, the resistance of in vivo net photosynthesis to Ni and Pb was correlated with the increased production of chloroplast small Hsp in a heat-tolerant selection genotype, compared to a near-isogenic heat-sensitive progenitor genotype making less chloroplast small Hsp (Fig. 5). These results indicate that the chloroplast small Hsp can protect photosynthesis during heavy metal stress. Thus, this study provides some of the first evidence that Hsps of any kind are involved in protecting photosynthesis during heavy metal stress. Interestingly, in corn, purified small Hsp did not protect Phet from Ni in vitro, but in A. stolonifera, protection of Phn was indicated, supporting the prediction that small Hsp could protect other aspects of chloroplast function from heavy metals besides Phet, such as Calvin cycle enzymes (including rubisco), which are known to be readily damaged by excess heavy metals (Stiborova et al., 1986
; Lidon and Henriques, 1991
; Angelov et al., 1993
; Moustakas et al., 1994
).
Production of chloroplast small Hsp was induced (Fig. 2) even before damage to photosynthesis was evident (e.g., day 4 at 500 and 1000 µmol/L metal) (Fig. 1). In addition, the results suggest the possibility that at lower metal levels, chloroplast smHsp accumulation increases with metal accumulation in leaves, while at intermediate levels of metals, smHsp accumulation saturates, prior to declining at high levels of metals, that likely are so toxic that even smHsp production is inhibited. Together, the results indicate that production of chloroplast small Hsp is an early response to heavy metal accumulation in leaves and that the function of chloroplast small Hsp is to limit damage to photosynthesis, rather than involvement in repair or recovery from heavy metal damage (a similar role is suggested for heat stress; Waters et al., 1996
; Downs et al., 1999a
; Török et al., 2001
). The ability of chloroplast small Hsp to protect photosynthesis from heavy metals was significant, both in vitro and in vivo, suggesting the potential utility of breeding, engineering, or selecting plants for increased production of chloroplast small Hsp (e.g., either constitutive production, more rapid induction, or increased accumulation during stress) in improving plant tolerance to heavy metals. Of course, increased production of the chloroplast small Hsp in response to heavy metal accumulation is only one of the many plant adaptive responses to heavy metals. Given that heavy metals damage a wide range of cell components and functions, significant increases in the tolerance of whole plants to heavy metals likely requires changes in multiple traits.
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FOOTNOTES |
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4 scott.heckathorn{at}utoledo.edu
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Angelov M. T. Tsonev A. Uzunova K. Gaidardjieva 1993 Cu2+ effect upon photosynthesis, chloroplast structure, RNA and protein synthesis of pea plants. Photosynthetica 28: 341-350[ISI]
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