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Plant Physiology 132:979-987 (2003) © 2003 American Society of Plant Biologists Physiological and Molecular Assessment of Altered Expression of Hsc70-1 in Arabidopsis. Evidence for Pleiotropic Consequences1Plant Molecular and Cellular Biology Program, Department of Environmental Horticulture, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611–0670
Hsp70s function as molecular chaperones. The protective chaperone activities of hsp70 help to confer tolerance to heat, glucose deprivation, and drought. Overexpression of hsp70s in many organisms correlates with enhanced thermotolerance, altered growth, and development. To better understand the roles of hsp70 proteins in Arabidopsis, the molecular and physiological consequences of altered expression of the major heat shock cognate, Hsc70-1, were analyzed. Extensive efforts to achieve underexpression of Hsc70-1 mRNA using a full-length antisense cDNA resulted in no viable transgenic plants, suggesting that reduced expression is lethal. Constitutive overexpression of Hsc70-1 also appeared to be deleterious to viability, growth, and development because fewer transformants were recovered, and most were dwarfed with altered root systems. Despite being dwarfed, the overexpression plants progressed normally through four selected developmental stages. Heat treatment revealed that Hsc70-1 overexpression plants were more tolerant to heat shock (44°C for 10 min). The elevated basal levels of HSC70-1 in transgenic plants led to delayed heat shock response of several heat shock genes. The data in this study suggest that tight regulation of Hsc70-1 expression is critical for the viability of Arabidopsis and that the functions of HSC70-1 contribute to optimum growth, development, thermotolerance, and regulation of the heat shock response.
Hsp70 functions as a molecular chaperone by helping newly synthesized proteins fold properly, preventing unfolded proteins from undergoing nonproductive aggregation and maintaining an extended conformation of proteins during translocation (Feldman and Frydman, 2000  ; Sung et al., 2001a). As a component of protein import mechanisms, hsp70 facilitates import of precursor proteins by establishing directional movement into the organelles (Matouschek et al., 2000; Jackson-Constan et al., 2001). These chaperone functions may involve not only the repetitive cycles of binding and the release of substrate peptides but also the active twisting of peptide bonds because it operates as a secondary amide peptide bond cis-trans isomerase (Schiene-Fischer et al., 2002).
Molecular chaperone functions of hsp70 are important in thermotolerance. The expression of hsp70 genes positively correlates with the acquisition of thermotolerance (Feder et al., 1996
Hsp70 is involved in regulation of the general heat shock response. Interaction between hsp70 and HSF has been suggested as a negative regulatory mechanism for HSF-mediated transcriptional activation before, after, and during the heat shock response (Morimoto, 1998
Successful alteration of hsp70 expression level in plants has been reported. Lee and Schöffl (1996
Arabidopsis has five full-length cytosolic hsp70s and a sixth that is truncated in the C-terminal domain (Lin et al., 2001
Previously, we assessed functions of hsp70s in Arabidopsis by analyzing expression patterns of the hsp70 gene family using a quantitative reverse transcriptase (RT)-PCR analysis (Sung et al., 2001b
Plant Transformation Plants were transformed with four different constructs (Fig. 1). PCR screening for primary transformants revealed that the frequency of transformation is distinct for each construct (Table I). Empty vector was transformed with an efficiency of 0.62%. However, no primary transformants for constitutive underexpression of Hsc70-1 were obtained after extensive kanamycin screening. This suggests that constitutive expression of a full-length Hsc70-1 antisense RNA may be lethal. The inducible overexpression construct, however, showed an equal efficiency to the empty vector control (Table I). This suggests that the harmful effects of the constitutive overexpression construct resulted from ectopic expression of Hsc70-1, not from a cosuppression mechanism due to the presence of the Hsc70-1 construct in the genome. In addition, low transformation efficiency for the constitutive overexpression construct (approximately 30 times lower compared with the empty vector construct) indicates overexpression of Hsc70-1 is also detrimental. Inducible overexpression/underexpression of Hsc70-1 was attempted by using a cold-inducible promoter (Cor78/Rd29A). Transformants for inducible overexpression were easily obtained with transformation efficiency comparable with that of the empty vector (Table I). However, growing plants at low temperature (4°C) to induce the transgene expression complicated plant growth and development, making assessment of phenotypic consequences of inducible expression of transgenes unreliable. A search for a more suitable inducible system is currently under way.
A modified adaptor ligation PCR technique was used to confirm integration of T-DNA into the Arabidopsis genome as an alternative to Southern-blot analysis (Spertini et al., 1999
Western-blot analysis of eight Hsc70-1 OE lines identified several lines containing a higher level of HSC70-1 protein compared with empty vector-transformed plants (pHK; Fig. 3). Overexpression lines 8-2, 8-4, 8-9, and 8-10 showed an approximately 4-fold increase in the amount of protein. Line 8-7 had the highest level of protein, 8-fold compared with wild-type plants. Lines 8-3, 8-5, and 8-11 showed little or no difference in the amount of protein compared with empty vector-transformed plants.
Four strong Hsc70-1 OE lines (8-4, 8-7, 8-9, and 8-10) were selected for further analysis. Thermotolerance of the four lines was tested by giving a 10-min heat shock in water at temperatures ranging from 42°C to 48°C. Only the samples treated with room temperature, 44°C, and 46°C were shown in Figure 4B. These four lines showed lower electrolyte leakage and survived a 10-min heat shock at 44°C (Fig. 4, A and B). Empty vector-transformed plants showed greater electrolyte leakage and showed no sign of survival 3 d after heat shock (Fig. 4B). At the temperatures below or above 44°C, no difference in thermotolerance was observed in the four overexpression lines compared with empty vector-transformed plants (Fig. 4, A and B).
It was noticed during screening of primary transformants that numerous kanamycin-resistant seedlings of Hsc70-1 OE plants had shorter and more branched root systems than empty vector-transformed plants (pHK). To recapitulate this phenomenon without kanamycin treatment, seeds of the overexpression lines were planted on agar plates and grown vertically to monitor root growth and development. One week after growing on vertical plates, root length was measured. The length of the primary root was reduced in Hsc70-1 OE plants compared with empty vector-transformed plants (Fig. 5). Also, the overexpression lines showed early branching of lateral roots (Fig. 5A). Early lateral root formation primarily occurred in the upper part of the root system (Fig. 5A). The identical results were obtained when seedlings were grown on horizontal Murashige and Skoog media indicating that alterations of root system in the overexpression lines are robust (Fig. 5C).
In addition to short and branched root systems, the Hsc70-1 OE plants had significantly smaller aerial parts (leaf, stem, and flower) compared with empty vector-transformed plants (Fig. 6). However, this difference only became apparent at 4 weeks after imbibition (Fig. 6B). These alterations in growth and development appear specific to Hsc70-1 because overexpression of an ER luminal hsc70, BiP-2 (data not shown), and Hsp101 (Queitsch et al., 2000
The overexpression plants have smaller stature compared with empty vector-transformed plants (Fig. 7A). However, the number of days to reach each of four selected stages was indistinguishable from the empty vector-transformed plants showing the aerial parts of Hsc70-1 OE plants, and empty vector-transformed plants progressed through different stages of development at the same rate (Fig. 7B). To test whether smaller plant size resulted from any defects in electron transfer of photosynthesis, chlorophyll fluorescence was analyzed with the same plants shown in Figure 6A. Almost identical values of maximum photochemical efficiency of PSII in the dark-adapted state (0.86 ± 0.01, n = 18–20) were obtained from the empty vector-transformed plants and Hsc70-1 OE plants, indicating electron transfer of photosynthesis in Hsc70-1 OE plants was not compromised.
To analyze the effect of Hsc70-1 overexpression on other closely related heat shock genes, the expression of five cytosolic members of the hsp70 gene family and Hsp101 was monitored in Hsc70-1 OE plants. All five members showed maximum induction within 60 min of heat treatment at 40°C (Fig. 8A). Two significant differences between the empty vector-transformed plants (pHK) and Hsc70-1 OE plants (8-7) were observed. First, Hsc70-1 OE plants showed moderately elevated expression of four other cytosolic hsp70 genes and Hsp101 in the absence of heat shock (Fig. 8A). Second, the induction after 10 min of heat treatment at 40°C was reduced in Hsc70-1 OE plants compared with the empty vector-transformed plants (Fig. 8A). The expression of Hsp101 was further analyzed with more time points during the first 30 min of heat treatment to study the induction kinetics of the heat shock response in Hsc70-1 OE plants in detail. Total RNA samples from empty vector-transformed plants and another Hsc70-1 OE line (8-10) were isolated at 0, 5, 10, 15, 20, and 30 min at 40°C and subjected to RT-PCR analysis. The expression of Hsp101 in empty vector-transformed plants (pHK) was strongly induced even after 5 min at 40°C and reached peak expression between 20 and 30 min at 40°C (Fig. 8B). However, the induction of Hsp101 was slower in the Hsc70-1 OE plant (8-10), but the time required to reach the peak expression remained unchanged (Fig. 8B).
Maintenance of the Level of HSC70-1 in the Cell Is Critical to Cell Viability
Transformation of pHK-Hsc70-1 (AS) yielded no viable transformants. This suggests that constitutive antisensing of Hsc70-1 alone or, alternatively, that global suppression of all cytosolic members resulted in the lethal phenotype. We have identified several hsp70 T-DNA insertion mutants, including one for Hsc70-1 (SALK_135531) in the Salk T-DNA insertion stocks. T-DNA in SALK_135531 is inserted in the second exon of Hsc70-1 and should render the gene product nonfunctional. Seed for SALK_135531 is not available yet. Once we obtain homozygous lines (or inability to obtain homozygous lines) for SALK_135531, we can address whether the knockout of Hsc70-1 alone is sufficient to render a lethal phenotype. Considering that the transformation constructs were generated using a full-length cDNA of Hsc70-1 encoding highly conserved domains of all hsp70s (Sung et al., 2001a
Constitutive overexpression of Hsc70-1 appears to be deleterious to plant viability given the lower transformation efficiency compared with that of the empty vector control. Similar findings have been obtained from overexpression of bacterial hsc70, DnaK, in Escherichia coli (Blum et al., 1992
Thermotolerance is a quantitative trait (Ottaviano et al., 1991
Altered root systems and smaller aerial parts of the overexpression plants suggest HSC70-1 plays important roles in plant growth and development. The modification of the root system in the overexpression plants indicates potential perturbation of hormonal signal transduction or nutrient utilization. Enhanced lateral root formation is frequently observed when Arabidopsis is grown in a low-phosphate or a high-nitrate environment (Zhang and Forde, 2000
A delayed heat shock response was observed in the expression pattern of Hsp101 and five hsp70 genes in overexpression lines. This suggests that the delay of the heat shock response did not result from a sequence-based cosuppression (i.e. gene silencing) limited to Hsc70-1-related genes but resulted from alteration of a common regulatory mechanism (likely HSFs). The repression of the heat shock response by hsp70 has been shown in overexpression studies of mammalian hsp70 homologs (Mosser et al., 1993
If the delayed heat shock response indeed resulted from the prolonged interaction between HSC70-1 and HSF, one puzzling question is: "How was the elevated basal expression of other heat shock genes possible if HSF was tied up with HSC70-1 in the absence of heat shock?" A subfamily of HSFs (HsfBs) in Arabidopsis has no transactivation activity. HsfBs are suspected to act as transcriptional repressors by occupying HSEs in the promoter (Czarnecka-Verner et al., 2000
Constitutive overexpression of Hsc70-1 resulted in several distinct phenotypes. At minimum, this study confirmed that altered expression of a single hsp70 gene could penetrate the potentially highly redundant functional background of cytosolic hsp70s and produce phenotypic consequences. Finding no viable transgenic lines for constitutive underexpression of Hsc70-1 suggests that reduction of Hsc70-1 expression is detrimental to plant viability. Constitutive overexpression of Hsc70-1 enhanced basal thermotolerance, affected plant size, and altered the root system. The overexpression also delayed the heat shock response of several heat-inducible genes probably through prolonged interaction with Arabidopsis HSFs. This study revealed that HSC70-1 is linked with enhanced thermotolerance and influences regulatory functions on the expression of multiple genes during heat stress. The small stature and altered root system of Hsc70-1 overexpression plants revealed that the functioning of HSC70-1 is pivotal in normal growth and development under non-stress conditions.
Plant Growth Arabidopsis (ecotype Columbia) plants were grown on an autoclaved commercial soil mix (Fafard mix no. 2) containing Canadian sphagnum peat, perlite, and vermiculite. Seeds were sown through holes made in the lids of clear delicatessen containers (a gift from Publix Super Markets Inc., Lakeland, FL). Plants grew better in this container due to fewer impediments on plant growth from fungus gnats and mold on the soil. It was also convenient to administer heat treatment where only the aerial portion of whole plants was immersed in a water bath. Plants were grown at 20°C with a photoperiod of 15 h of light/9 h of dark in Percival growth cabinets (Percival Scientific, Inc., Perry, IA). Irradiance was approximately 150 µmol m-2 s-1 at canopy height and was provided by incandescent bulbs and cool-white fluorescent tubes.
The proprietary transformation vector (pHK1001) obtained from Dr. Harry Klee with consent from Monsanto Co. (St. Louis) was used to generate Hsc70-1 constitutive and inducible expression constructs (Fig. 1). A full-length expressed sequence tag (G9H1T7) for Hsc70-1 was used to prepare the constructs. The final constructs were then transferred to an Agrobacterium tumefaciens strain (ABI) by a triparental mating method. Escherichia coli (DH5
Putative transformants obtained after kanamycin selection were subjected to PCR screening. Primers specific to promoters (figwort mosaic virus) and Hsc70-1 were designed to amplify the segments of the transgenes in kanamycin-resistant plants. A primer pair for each promoter and construct produced a distinct-sized PCR product. Genomic DNA from each transformant was extracted as described (Li and Chory, 1998
Genomic DNA (1 µg) was digested with a 4-bp restriction enzyme, TaqI, to generate smaller restriction fragments. Two adaptor primers (CG336, 5'-ctaatacgactcactatagggctcgagcggccgggcaggt-3'; and CG337, 5'-gcacctgcccaa-3') were ligated by heating the primer solution at 80°C for 2 min followed by a stepwise cool down over 40 min to produce the adaptor that ligates to TaqI digested genomic DNA. Ligation of genomic DNA and the adaptor was carried out as described (Spertini et al., 1999
Total cell extract was prepared by grinding fresh leaf samples from 4-week-old plant in 2x SDS-loading buffer (plant sample:buffer, 1:3 [w/v]). The supernatant obtained after centrifugation at 5,000g for 5 min was separated by SDS-PAGE. The proteins were then transferred onto a polyvinylidene difluoride membrane using a semidry blot transfer cell (Transblot SD, Bio-Rad Laboratories, Hercules, CA) and immunoreacted with a monoclonal antibody raised against spinach (Spinacia oleracea) cytosolic hsc70 (SPA-817, Stressgen, Victoria, BC, Canada). The same polyvinylidene difluoride membrane was cut in the middle and incubated with a polyclonal antibody (a gift from Dr. Kenneth C. Cline, University of Florida, Gainesville, FL) against an LHCP and then simultaneously developed as a loading control. The antibody used for HSC70-1 is specific to cytosolic hsp70 proteins; however, it has not been established whether this antibody reacts specifically to HSC70-1 or to several or all cytosolic hsp70 proteins.
Tissue samples were ground in liquid nitrogen, and total RNA was isolated according to the manufacturer's protocol using the RNeasy plant mini-kit (Qiagen USA, Valencia, CA). The amount of total RNA was determined by UV spectrophotometry. Using commercial RT-PCR beads (Amersham Biosciences, Piscataway, NJ), aliquots of total RNA were reverse transcribed into cDNA with 0.5 µg of random primer, d(N)6, by incubating the reactions at 42°C for 30 min, then amplified with gene-specific primers (10 pmol each) in a 25-µL reaction through 25 cycles of PCR. For each RT-PCR reaction, primers for an 18S rRNA internal standard were included as a loading control. 18S rRNA signals were adjusted by using either 3:7 mix (normal pair of primers: 3'-modified primer pairs) or 2:8 mix to best balance the 18S rRNA signal to that of the target mRNA. PCR parameters were as follows: 94°C for 4 min for initial denaturation, 25 cycles of 94°C for 30 s, 60°C for 1 min, 72°C for 2 min, and 72°C for 10 min for the final extension before holding the reaction at 6°C. The annealing temperature for Hsc70-2 was 52°C and 60°C for Hsc70-1 and all other genes analyzed in this study. The forward primer for Hsp101 is 5'-agcaatctctagtgccggtg-3', and the reverse primer is 5'-aagcgttgtagcaccaatgc-3'. Primers for Hsc70-1, Hsc70-2, Hsc70-3, Hsp70, and Hsp70b were previously described (Sung et al., 2001b
Thermotolerance assays were conducted by inverting the containers over water baths allowing the aerial portion of 4-week-old plants to be immersed to the soil line at temperatures of 42°C, 44°C, 46°C, 48°C, and 50°C for 10 min. Electrolyte leakage of the aerial portion of a plant was measured 3 d after heat treatment. The aerial portion of a plant was cut and immersed in a vial with 10 mL of distilled, deionized water. Samples were shaken for 1 h at room temperature, and ion leakage measurements were taken. Samples were then heated to boiling in a microwave for 2 min, shaken for 1 h at room temperature, and total ion leakage measurements were taken. With the two measurements, percentage of electrolyte leakage was determined for each sample. For leaf area assays, 5-week-old plants were digitally photographed and the aerial portion (in pixels) was quantified using Adobe Photoshop (Adobe Systems, San Jose, CA). The roots of plants vertically grown on Murashige and Skoog media for 2 weeks were measured for root growth. To monitor growth and development of plants, the number of days taken to reach four selected parameters (four true leaves, eight true leaves, first bolting, and first open flower) was recorded. Electron transfer in PSs was measured by recording chlorophyll fluorescence parameters with the Plant Efficiency Analyser (Hansatech Instruments, King's Lynn, UK). Leaves of 5-week-old plants were given a 10-min dark adaptation period, then measurements were taken over a 5-s interval after exposure at the 100% illumination level by high-intensity light-emitting diodes. Five replicates, each from a different plant, were averaged for each time point. Variable fluorescence was determined as the difference between the maximal fluorescence signal and the initial darkness fluorescence level.
We thank Dale Haskell for developing the thermotolerance assay system. We appreciate Drs. William B. Gurley, David Clark, and Kiljae Lee providing critical reviews of this manuscript. Special thanks go to Dr. Kenneth C. Cline for providing access to the gel documentation system and LHCP antibody and to the Arabidopsis Biological Resource Center for providing a full-length expressed sequence tag clone for Hsc70-1. Received December 18, 2002; returned for revision February 15, 2003; accepted March 6, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.102.019398.
1 This work was supported by the Florida Agricultural Experiment Station, by the University of Florida Plant Molecular and Cellular Biology Program, and by the U.S. Department of Agriculture National Research Initiative (grant nos. 98–35100–6147 and 00–35100–9532 to C.L.G.). This is Florida Agricultural Experiment Station Journal Series No. R–09359. * Corresponding author; fax 352–392–3870; e-mail clguy{at}ufl.edu.
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) with pRK2013 was used as the helper cell. For plant transformation, a modified version of the vacuum infiltration method was used (Bent et al., 1994
