INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plants, as other living organisms, respond to the increase of temperature with general changes in gene expression known as the heat shock response. Such response involves the transcriptional activation of genes encoding characteristic stress proteins: the heat shock proteins (HSPs; Cotto and Morimoto, 1999). The activation is mediated by conserved trans-acting factors and cis-acting elements known as the heat shock transcription factors (HSFs) and heat shock elements (HSEs), respectively. In addition, gene expression during heat shock is subjected to a strict post-transcriptional control that results in the translational arrest of most mRNAs and the preferential translation of the HSP encoding mRNAs. Such control has been shown to involve sequences located in the 5'-untranslated region (5'-UTR) of the HSP mRNAs (for plant genes, see Joshi and Nguyen, 1995, and refs. therein). In plants, the heat shock response is peculiar in the higher diversity of genes encoding HSPs with small (15-30 kD) molecular size, the small HSPs (sHSP; Waters et al., 1996). Such diversity, and the conservation among different members of the various classes of sHSPs, has complicated the analysis of their individual gene expression patterns. RNase A protection and nuclear run-on assays allowed gene-specific detection of sHSP mRNA accumulation and transcription (Carranco et al., 1997, and refs. therein). Using such techniques, we demonstrated that in sunflower (Helianthus annuus), there are at least two class I sHSP genes that are heat inducible in most vegetative tissues, i.e. the stem and leaves of adult plants, as well as in young seedlings: Ha hsp17.7 G4 and Ha hsp18.6 G2 (Coca et al., 1996; Carranco et al., 1997). Only Ha hsp17.7 G4 is also transcriptionally activated in seeds during embryogenesis at normal temperature (Carranco et al., 1997). Expression in seeds from the Ha hsp17.7 G4 promoter could be faithfully reproduced and analyzed in transgenic tobacco (Nicotiana tabacum) plants using chimeric genes (Coca et al., 1996; Almoguera et al., 1998). This contrasted with the results obtained for expression of the same chimeric genes in response to heat shock.

Analyses of the heat shock response in plants have been also attempted by the use of chimeric genes in transgenic systems. Different authors have used two types of gene fusions: transcriptional (Prändl et al., 1995) and, more frequently, translational fusions (Takahashi and Komeda, 1989; Takahashi et al., 1992; Coca et al., 1996; Moriwaki et al., 1999; Wehmeyer and Vierling, 2000). By including not only the promoter but also the complete 5'-UTR plus a short part of the open reading frame of a given HSP, expression of the chimeric reporter construct usually reflects all essential aspects of heat stress regulation (transcriptional and post-transcriptional). Most studies were performed using the -glucuronidase (GUS) reporter gene in heterologous host plants (Takahashi and Komeda, 1989; Coca et al., 1996; Moriwaki et al., 1999), and in the case of Arabidopsis genes, the homologous host was used (Takahashi et al., 1992; Wehmeyer and Vierling, 2000). A conclusion from most of these analyses is that, independently of the type of gene fusion and transgenic system, the heat shock response was faithfully reproduced. However, there were also exceptions. For example, some results seemingly showed specificity in the heat shock response. This has been the case of Ha hsp17.7 G4, one of the class I sHSP genes from sunflower analyzed in transgenic tobacco. Translational fusions of Ha hsp17.7 G4 showed that heat induction of GUS activity was only observed in stems and not in leaves or whole seedlings at early germination stages (Coca et al., 1996). These results contrasted the transcription (Carranco et al., 1997) and mRNA accumulation studies (Coca et al., 1996; Carranco et al., 1997) performed in sunflower and described above. This possible discrepancy could be explained by transcriptional or post-transcriptional, tissue-specific differences in Ha hsp17.7 G4 gene expression. Other intriguing results, using a translational fusion, have been reported for the At hsp18.2 gene. In these studies, the reporter GUS activity substantially increased after heat shock, during recovery at control temperature. Furthermore, immediately after heat shock, the induced GUS activity was almost absent (Moriwaki et al., 1999). These studies were performed in the Arabidopsis homologous host (Takahashi et al., 1992) or in transgenic tobacco (Moriwaki et al., 1999). The authors proposed different translational effects to explain such results (Takahashi et al., 1992; Moriwaki et al., 1999).

In this work, we compared chimeric mRNA and protein expression patterns obtained from the original Ha hsp17.7 G4 translational fusion and a derived transcriptional fusion in transgenic tobacco. Both contained the promoter, the same regulatory 5'-flanking sequences, and the complete 5'-UTR. The transcriptional fusion did not include protein-coding sequences from Ha hsp17.7 G4. We found that the choice of the chimeric gene did not affect the results of analyses of developmental regulation in seeds at control temperatures. However, the inclusion of the additional amino acid residues in the translational fusion abolished the heat-induced activity of the reporter gene in young seedlings. Surprisingly, this effect was post-translational because it involved the heat inactivation of the chimeric GUS protein activity. However, heat inactivation of the chimeric and non-chimeric GUS proteins was similar in vitro. In the transgenic plants, the inactivation of chimeric GUS was fully reversible after returning to normal growth temperatures. The inactive state of the chimeric protein correlated with its insolubilization, as inferred from immunoprecipitation experiments. We also observed a comparable inactivation and reactivation using a translational fusion containing the 21 first amino acids of Ha hsp18.6 G2, another sHSP from sunflower that belongs to class I (Coca et al., 1996). These results strongly indicate that in vivo inactivation of chimeric GUS proteins involves transient interaction(s) with cellular factor(s). Such interactions would require conserved sequences within the first 20 amino acids of class I sHSPs and would result in the transient insolubilization of the chimeric proteins in vivo.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The Inclusion of Additional Amino Acids in the 1,132::GUS (WT) Chimeric Gene Does Not Affect Expression in Seeds at Control Temperatures

We previously reported the use of translational fusions to the GUS gene that contain the promoter and 5'-flanking sequences of Ha hsp17.7 G4. One such fusion (WT) reproduced in transgenic tobacco the transcriptional activation and mRNA accumulation patterns also observed in sunflower embryos (Coca et al., 1996; Carranco et al., 1997). Its heat shock response was, however, apparently almost absent in most vegetative tissues (Coca et al., 1996). To investigate potential effects of the Ha hsp17.7 G4-coding sequences in the WT translational fusion, we constructed a similar chimeric gene (BglII), in which these sequences were deleted (see Fig. 1 and "Materials and Methods" for details). The original WT chimeric gene is a translational fusion containing the first 85 nucleotides of the Ha hsp17.7 G4 protein-coding sequence fused in frame to GUS (Coca et al., 1996). WT, thus, encodes a chimeric GUS protein that contains the 28 amino-terminal amino acids from Hsp17.7 and seven amino acids encoded by synthetic linker sequences from the pBI 101 vector. In contrast to this, BglII is a transcriptional fusion encoding the bacterial GUS protein without additional amino acid residues.

View larger version (18K): [in this window] [in a new window]   Figure 1.   Top, Map of the WT (Coca et al., 1996) translational fusion. Proximal (I) and distal (II) HSE regions are indicated as small boxes and transcription initiation with an arrow. The Ha hsp17.7 G4-coding sequences are shown as a large black box. Bottom, Map of the BglII transcriptional fusion. Below the maps, The nucleotide sequences between position +163 (WT) or +79 (BglII) and the first amino acid in the GUS protein (ATG). In each case, the pBI 101.1 sequence before the GUS ATG is shown, with small differences resulting from cloning indicated in lowercase (see details in "Materials and Methods"). For WT, the additional amino acids encoded by vector sequences are shown in brackets.

We produced 13 different primary (T0) tobacco transgenic plants for the new BglII chimeric gene and selected five plants showing single integration events. As a first step in our investigation, we verified the expression patterns of the WT and BglII genes in seeds that were collected after growth and development at a normal temperature. We performed quantitative fluorometric GUS activity determinations and evaluated levels of chimeric mRNA accumulation. As an internal standard for mRNA accumulation, we used the neomycin phosphotransferase transcripts encoded by the binary vector. We compared the samples from BglII plants with those of three representative WT plants obtained previously (Coca et al., 1996). An arbitrary value of 100 was assigned for both the GUS activity and the standardized mRNA band intensity of the WT samples. Normalized GUS activity was defined as the ratio between both values and, thus, was equal to one for the WT gene.

The results from such experiments are shown in Figure 2. In seeds compared with the WT chimeric gene, the BglII gene was expressed to similar levels of GUS activity (Fig. 2A) and mRNA accumulation (Fig. 2B). The normalized GUS activities for both genes were virtually identical (Fig. 2C). Thus, a slight increase in mRNA accumulation for the BglII plants corresponded to a similar increase of GUS activity. This indicates that the protein expression levels from both chimeric genes are very similar. We could not directly verify this because of technical problems with the detection of GUS using antibodies against GUS in the seed samples. However, other experiments indicated that the different GUS proteins, encoded by the WT and BglII genes, showed similar stability at control temperatures in seedlings. Even seed extracts containing such proteins showed comparable heat-inactivation profiles (see below). Thus, we conclude that at least in seeds during development at normal temperature, the differences between the WT and BglII genes were irrelevant for gene regulation.

View larger version (57K): [in this window] [in a new window]   Figure 2.   GUS activity and chimeric mRNA accumulation, for the WT and BglII genes, in seeds. We show the results of two independent experiments (1 and 2) performed with seeds from the different transgenic plants collected at 24 (1) or between 24 and 28 DPA (2). For both GUS and RNA assays, seed samples representing each gene (two seedpod per individual transgenic plant) were pooled, homogenized, and frozen. GUS assays were performed in duplicate with extracts prepared from small aliquots of the frozen-pooled samples. The averages GUS activities obtained for each gene are shown in A. The rest of each sample was used for RNA preparation and RNase A protection experiments. The RNase protection results from experiments 1 and 2 are separately presented in B. The results shown were obtained using either the chimeric Ha hsp17.7 G4::GUS (hsp17.7::GUS; top) or the neomycin phosphotransferase (NPTII, internal control; bottom) antisense riboprobes. M, Size marker; HpaII digested, pBluescript SK, labeled DNA. P, Undigested probes. In C, we summarize the relative values of mRNA accumulation (mRNA*), GUS activity (GUS*), and normalized GUS activity (GUS*/mRNA*) obtained from both experiments as explained in the text.

Heat Shock Induced the Reversible Inactivation of the Chimeric GUS Protein. Effects on the Detection of Chimeric Gene Expression

Previous analyses of the heat shock response of Ha hsp17.7 G4, using the WT gene and other chimeric genes derived from this construct, apparently did not faithfully reproduce the response to heat shock observed with the Ha hsp17.7 G4 gene in sunflower (Coca et al., 1996; Carranco et al., 1997). In particular, we observed a discrepancy between the strong transcriptional activation of the Ha hsp17.7 G4 promoter in the nuclei of sunflower seedlings (Carranco et al., 1997) and the absence of heat-induced GUS activity when the WT chimeric gene was expressed in transgenic seedlings (Coca et al., 1996). We assumed that this difference could be explained by post-transcriptional events. To address this question, we compared the heat-induced expression patterns of the WT and BglII genes using tobacco seedlings. Our experimental design was analogous to the one used for seeds. We decided to employ young tobacco seedlings, because this material could be subjected to quick bulk analysis of both mRNA accumulation and GUS activity. In addition, the use of seedlings facilitated the immunological detection of the GUS proteins using commercial antibodies (Figs. 3 and 4).

View larger version (47K): [in this window] [in a new window]   Figure 3.   GUS activity and chimeric mRNA accumulation for the WT and BglII genes in seedlings. A, Fluorometrical determinations. We show average values obtained from one representative experiment, at control (C, 25°C) and after heat shock (HS, 42°C) conditions, performed with the progeny of different primary transgenic plants for the WT or BglII gene (see Fig. 1). Protein extracts were prepared from 25 seedlings per each primary plant and separately assayed. The ratio between the C and HS activities obtained in each case is indicated. Statistical significance of the observed differences is indicated by asterisk(s) (see text for statistic values). B, Chimeric mRNA accumulation level in samples from the same experiment shown in A. Seedlings for each chimeric gene were pooled for the preparation of the analyzed RNA samples. We show the results of RNase A protection experiments depicted as explained in the legend of Figure 2. C, Comparison of relative and normalized values for GUS activity and mRNA accumulation (symbols as in Fig. 2).

View larger version (83K): [in this window] [in a new window]   Figure 4.   A, Heat-induced accumulation of the GUS proteins immediately after stress treatment and during stress recovery. The proteins encoded by the WT and BglII genes were immunodetected with antibodies against bacterial GUS. Samples were analyzed under control temperature conditions (C), immediately after heat shock at 42°C (HS), or following recovery after heat shock. In this case, the heat-stressed seedlings were returned to incubators at 25°C for 3 (R3), 17 (R17 in B), and 24 h (R24) before extract preparation. The two arrows indicate the position of the proteins encoded by WT (top) and BglII (bottom). Note the size shift due to the addition of amino acid residues in the WT protein. The position of molecular mass markers (in kilodaltons) is indicated to the left. Asterisks mark abundant polypeptides present in the protein extracts that were detected as negative (white) signals. Detection in all samples as negative signals of the most abundant polypeptides in the extracts confirmed that equal amounts of protein were loaded, as was also confirmed by Ponceau-S staining of the membrane before reaction with the antibodies (data not shown). B, Recovery of GUS activity of the WT protein after heat shock. Mean GUS activity values for the WT and BglII genes correspond to the samples in A and to an intermediate stress recovery time (R17). Stress treatments and experimental sample size as described in the legend of Figure 3.

As reported earlier (Coca et al., 1996), low levels of GUS activity were detected in seedlings harboring the WT gene at control temperature (169.2 pmol methylumbelliferone mg¹ min¹), and they did not significantly increase upon heat shock (Fig. 3A; 1.6-fold, P = 0.41). In contrast to the results of enzyme activity, chimeric Ha hsp17.7 G4::GUS mRNA from the WT gene was not detectable at control temperature (Fig. 3B) but accumulated to a high level in response to heat stress, indicating that GUS activity is controlled by post-transcriptional mechanisms (Fig. 3, A and C).

Similar analyses performed with the BglII chimeric gene revealed significant heat-induced accumulation of mRNA, which correlated with a corresponding increase of GUS activity (79.5-fold, P = 0.0001; Fig. 3A). Comparison of normalized values of gene expression revealed that, based on the GUS activity, the BglII gene was 139-fold more active than the WT gene (Fig. 3C).

Because the WT gene encodes a chimeric protein that is different from the bacterial GUS enzyme encoded by the BglII construct, we analyzed the actual protein accumulation levels as compared with enzyme activities in heat stress and recovery experiments (Fig. 4). Figure 4A shows the results obtained by immunodetection of GUS in total extracts prepared from samples harvested at the indicated time points. Both transgenic tobacco seedlings containing the WT or the BglII genes showed heat shock-induced synthesis of the respective GUS proteins to similar levels (Fig. 4A). Thus, the failure to detect heat-induced GUS activity for WT protein is not the consequence of its defective synthesis, but it is rather the consequence of a reversible inactivation by heat shock of the WT but not the BglII protein (Fig. 4B). In the recovery period, GUS activities were detectable in samples from both types of seedlings and reached comparable levels 24 h after heat stress without detectable changes in the corresponding GUS protein levels (Fig. 4B).

WT and BglII GUS Proteins Are Similarly Sensitive to in Vitro Heat Inactivation

In transgenic plants, the WT chimeric protein was inactivated by heat stress in conditions that did not affect the BglII protein. This may reflect an intrinsic property of the WT protein (i.e. explained exclusively by the additional amino acids in the translational fusion) or, alternatively, the requirement of cellular factors related to the heat treatment. To discriminate between the two possibilities, we performed in vitro heat-inactivation experiments with protein extracts from seeds (Fig. 5) and seedlings (data not shown). Pretreatment of extracts containing active BglII and WT proteins for different times at 42°C, 52°C, and 65°C revealed similar inactivation profiles. Both proteins were fully inactivated after 15 min at 65°C but were unaffected at 42°C and slowly inactivated at 52°C (Fig. 5). These results suggest that the inactivation observed in transgenic plants cannot solely be explained by intrinsic characteristics of the chimeric protein. The reversible inactivation of the WT fusion protein evidently also needs in vivo conditions that are specific to the heat shock response.

View larger version (20K): [in this window] [in a new window]   Figure 5.   Effect of the temperature on the activity of GUS encoded by the WT and BglII chimeric genes. Activities were assayed in vitro with protein extracts prepared from seeds of the WT (squares) or BglII (circles) plants (see legend of Fig. 2). We show the effect of preincubation for different time as the percentage of the initial GUS activity obtained after incubation for each time at two temperatures: 42°C (black symbols) or 52°C (white symbols). Absolute values for the initial activities were comparable. Treatment of both extracts for 15 min at 65°C fully inactivated the WT and BglII proteins (striped symbols).

Transient Insolubilization of the Chimeric WT Protein in Extracts from the Transgenic Plants

We attempted immunoprecipitation to further investigate protein interactions that would explain the heat-induced inactivation of the WT protein. If feasible, these experiments could lead to coprecipitation of cellular proteins that may interact with WT resulting in the observed inactivation. We used the GUS antibodies and protein extracts corresponding to the WT (HS and 24R) and BglII (HS) samples of Figure 4, which were prepared in non-denaturing conditions (Krishna et al., 1997). We first verified whether the WT and BglII GUS proteins could be efficiently immunoprecipitated using commercial GUS antibodies and published procedures (Krishna et al., 1997). We analyzed the immunoprecipitated proteins in denaturing gels. We could efficiently immunoprecipitate the BglII protein and the active form of the WT protein (from extracts prepared with the 24R sample). The inactive form of the WT protein could not be efficiently precipitated from extracts prepared from samples taken immediately after heat shock (Fig. 6). From these results we conclude that a reversible insolubilization of the WT protein could explain its inactivation and reactivation in heat-stressed transgenic plants. This insolubilization would be consistent with the proposed protein interactions, and with the in vivo observation of structure-bound complexes containing plant sHSPs (Kirschner et al., 2000; see "Discussion").

View larger version (93K): [in this window] [in a new window]   Figure 6.   Immunoprecipitation of the WT and BglII GUS proteins. The gels depict soluble proteins precipitated from extracts prepared under non-denaturing conditions from plant material corresponding to the experiment in Figure 4. Denominations are as in the legend of Figure 4. The arrow points to the immunoprecipitated GUS protein observed only in samples with GUS activity (WT 24R and BglII HS). The asterisk marks the band corresponding to the antibody heavy chain (55 kD). Each gel shows the result of independent experiments, with different precipitation efficiency in each case. Extracts prepared in denaturing buffer conditions from the same plant material confirmed that each sample contained comparable total amount of the GUS proteins (see Fig. 4B). Size of molecular mass markers in kilodaltons (left side).

Reversible Inactivation of a Different Chimeric GUS Protein Containing Similar sHSP Sequences

To explore the possibility that the cellular inactivation phenomenon results from the addition of the N-terminal 28 amino acid residues of the Ha hsp17.7 G4 protein, we tested the behavior of a similar fusion protein with a N-terminal tag derived from the related Ha hsp18.6 G2 protein (Coca et al., 1996). Considering GUS activity, the expression of this fusion was clearly heat inducible in transgenic tobacco seedlings (Fig. 7A). However, similar to the Ha hsp17.7 G4 fusion protein, a dramatic increase in GUS activity was only observed after recovery from heat stress (Fig. 7A). The analysis of GUS protein accumulation for the Ha hsp18.6 G2 fusion showed: (a) basically unchanged levels immediately after the heat stress treatment and at various times during the recovery process (Fig. 7B); and (b) much higher heat-induced levels of protein accumulation, compared with the previously analyzed WT protein as indicated by the different intensity of the bands detected by the antibody in Figures 4A and 7B, also consistent with the difference in GUS activities observed with each protein after recovery (Figs. 4B and 7A). These results support our interpretation that the observed reversible inactivation in vivo results from transient interactions involving the chimeric proteins and other cellular factors. Conserved sequences in the amino-terminal region of class I sHSPs may be involved in such interactions.

View larger version (82K): [in this window] [in a new window]   Figure 7.   Reversible inactivation of the Ha hsp18.6 G2 chimeric GUS protein after heat shock. A, Fluorometric quantification of GUS activity in control (C) and heat-induced (HS) transgenic seedlings and of activities obtained after allowing recovery for 3 to 48 h after heat shock (R3-R48). Experimental conditions and rest of symbols are as in Figures 3A and 4B. Experiments were performed with the progeny of three different transgenic lines. B, Western blot showing GUS protein accumulation corresponding to the samples analyzed in A (see the legend of Fig. 4A for other details).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Relevance of the Inclusion of Coding Sequences for Analyzing the Heat-Induced Expression of Plant sHSP Genes

The regulation of plant sHSP promoters has been analyzed mainly using fusions to the GUS reporter gene (Takahashi et al., 1992; Prändl et al., 1995; Coca et al., 1996; Moriwaki et al., 1999; Wehmeyer and Vierling, 2000). Previous studies of chimeric gene expression in transgenic plants assumed that the inclusion of sHSP-coding sequences as translational fusions did not affect reporter gene detection (Takahashi et al., 1992). We showed that the coding sequences included in the WT fusion (the sHSP- and linker-encoded additional amino acids; see Figs. 1 and 8) inactivated the heat-induced GUS activity of the chimeric protein in vivo (see Figs. 3 and 4). Thus, we found a very specific effect that would depend on peculiar protein sequences, on heat shock, and, perhaps most surprisingly, on the occurrence of in vivo conditions (see the results in Figs. 3 and 4 as opposed to the in vitro results in Fig. 5). The observed effect did not involve substantial changes in chimeric mRNA or protein accumulation; moreover, the effect was fully reversible after heat shock. Thus, the heat-induced expression patterns of the WT chimeric gene in tobacco matched those observed in sunflower (Coca et al., 1996; Carranco et al., 1997) but only when analyzed at the RNA or protein accumulation levels or after allowing time for recovery of GUS activity. We have found that the inclusion of the 5'-UTR sequences as either a transcriptional or a translational fusion is necessary for the efficient expression of the chimeric genes not only after heat shock, but also during embryogenesis at normal temperature (C. Almoguera, A. Rojas, and J. Jordano, unpublished data). Whereas the analysis of the developmental regulation is not affected by the type of fusion genes (Fig. 2), we conclude that allowing recovery from heat stress is perhaps the easiest and safest way to analyze the heat induction in transgenic plants of similar translational fusions.

View larger version (20K): [in this window] [in a new window]   Figure 8.   N-Terminal sequences of three class I sHSPs that here or in the literature (see text for details) have been analyzed as translational fusions to the GUS gene in transgenic tobacco plants. Left, The sHSP amino acids present in each fusion are shown (uppercase). Numbers below the sequences indicate position. Right, Additional amino acids (lowercase), encoded by plasmid linker sequences, present before the first Met of GUS (M). Protein denominations: At, Arabidopsis; Ha, Helianthus annuus. The mostly conserved 20 sHSP amino acids present in all fusions (left) and the only four amino acids conserved in the linker-encoded sequences (right) are underlined.

Reversible Inactivation of the WT GUS Protein in Vivo. A General Phenomenon Involving Conserved Amino-Terminal sHSP Sequences and Protein Interactions Specific to Heat Stress Conditions?

We propose that the inactivation and re-activation of the WT protein may be the consequence of in vivo protein-protein interactions. Such interactions would be transient and peculiar of heat shock conditions. By performing in vitro inactivation experiments, we determined that the observed inactivation did not depend solely on properties intrinsic to the chimeric protein (Fig. 5). The results of such experiments agree with reported high values for the heat stability of the GUS enzyme (T_1/2 at 55°C = 2 h) and its wide tolerance to N-terminal fusions (Jefferson, 1987). Such properties made GUS an appropriate choice as reporter for chimeric gene expression in plants at the temperatures normally used for heat shock experiments (35°C-42°C). As a consequence, heat inactivation of chimeric GUS proteins was totally unexpected. Extrinsic factors (i.e. interactions with cellular proteins), thus, would be required for the inactivation of the chimeric WT protein. This hypothesis is reinforced by the results from immunoprecipitation experiments (Fig. 6) and by those obtained with a different chimeric GUS protein (Fig. 7). In the latter case, we should compare protein accumulation and GUS activity levels observed for both chimeric proteins immediately after heat shock. Although in the case of the WT protein, lower protein accumulation levels (Fig. 4A) correlate with undetectable GUS activity (Fig. 4B), the second protein was expressed at much higher accumulation level and with higher activity (Fig. 7). Thus, the higher accumulation level of chimeric protein could exceed the proposed protein interactions. In the case of the WT protein, their lower accumulation results in excess of inactivating interactions and, thus, in its complete inactivation after heat stress (Fig. 4). Alternative explanations would require a differential and direct effect on GUS activity of the amino acids fused to GUS in each case. This is very unlikely given the absence of effect of such amino acids in the case of the inactivated WT protein (see Fig. 5). In the case of immunoprecipitation (Fig. 6), although these experiments could not be used to detect soluble cellular interacting proteins, we concluded that the inactive WT protein should be insoluble and that reversion of GUS activity corresponded with re-solubilization of this protein. These solubilization changes are indicative of in vivo transient interactions with unidentified cellular factors, most likely proteins. Recent results from our lab suggest that non-denaturing detergents, such as sodium deoxycholate up to a concentration of 2% (w/v), cannot reactivate the inactive protein in vitro (data not shown).

sHSPs oligomerize and interact as chaperones with model substrates (Lee et al., 1997), other cochaperones (i.e. from the HSP70 family; Forreiter et al., 1997; Lee and Vierling, 2000), and denatured cellular proteins (Jinn et al., 1995; Young et al., 1999). These interactions are largely unexplored in plant cells (for example, see Jinn et al., 1995; Lee and Vierling, 2000). Studies of in vivo interactions are even scarcer (Forreiter et al., 1997; Kirschner et al., 2000). We should note that the insolubility and detergent resistance characteristics of the inactive WT protein would fit with its transient association to HSPs in heat shock granules. These granules are produced in heat-stressed plant cells in vivo, showing large structure-bound aggregated forms that are detergent resistant (Kirschner et al., 2000).

The observation of the reversible inactivation with two different chimeric GUS proteins (Figs. 4 and 7) would also allow us pointing the significance of the conserved sHSP sequences present in these proteins. This comparison could include a third chimeric protein that also has been analyzed for heat-induction activity in transgenic tobacco (Moriwaki et al., 1999). This protein contains the first 26 amino acids of Arabidopsis Hsp18.2 and linker-encoded amino acids that differ from those in the two proteins analyzed here (Fig. 8). A delayed recovery of GUS activity after heat stress has been observed with the Hsp18.2 chimeric protein (Moriwaki et al., 1999). This could be re-interpreted to be indicative of reversible heat inactivation, at least in light of our results. The inclusion of this protein in our sequence comparisons would reinforce the significance of the 20 amino-terminal sHSP residues that are mostly conserved in the three chimeric proteins. That inclusion also reduced the possible contribution of other linker-encoded amino acids to only four residues (Fig. 8). We think that a major contribution of these four amino acids would be very unlikely. We should note that the conserved sHSP residues could have a disordered structure and be buried in the central hollow of the oligomeric sHSP complex. This may be inferred from crystallographic and fluorescence data of non-plant sHSPs (see Kim et al., 1998, and refs. therein). Thus, the possible localization of this region would fit with the hypothetical interactions with substrates or cochaperones discussed above. The pointed sHSP amino acid residues (Fig. 8) are also mostly conserved in other class I sHSPs (Waters, 1995). Therefore, reversible inactivation of chimeric GUS proteins may also be observed using similar sequences from other plant sHSP genes. The level of heat-induced inactivation reached in vivo in each case could depend on protein expression levels (i.e. a titration effect). That the inactivation level could also depend on slight sequence differences in the conserved sHSP region between the different proteins cannot be excluded. In conclusion, our observations would allow a more efficient choice of chimeric genes and experimental procedures for analyzing their shock response. We also open the possibility of using sHSP::GUS translational fusions as a tool for the investigation of a so-far elusive subject: in vivo protein interactions specific of heat stress conditions and involving plant sHSPs. This new tool may help to define functional distinctions among different plant sHSPs and to find crucial interactions for plant thermotolerance.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Chimeric Gene Construction and Production of Tobacco (Nicotiana tabacum) Transgenic Plants

The new transcriptional fusion BglII corresponds to an internal deletion in the Ha hsp17.7 G4 protein-coding sequences present in the previously described translational fusion WT constructed in the binary vector pBI 101.1 (Coca et al., 1996). WT and BglII, thus, contained Ha hsp17.7 G4 sequences between +1 (transcription start) and 1,132 and the complete 5'-UTR (nucleotides between +1 and +78). WT also contained Ha hsp17.7 G4 protein-coding sequences between the ATG (+79) and +163. To construct BglII, the Ha hsp17.7 G4 5'-flanking sequences upstream of the ATG were amplified from an appropriate plasmid using Pfu DNA polymerase (Stratagene, La Jolla, CA) and the primer 5'-CTTGGAATGATaGAtcTGGTTGGTAAC-3', which corresponds to the G4 non-coding strand. Thus, the ATG (bold) was mutated to a BglII restriction site (underlined, three nucleotide substitutions in lowercase). The PCR amplified DNA was cloned between the XbaI and BamHI sites of pUC19, and its nucleotide sequence was verified. These sequences were fused to GUS at the SmaI site of pBI 101.1, using an SmaI site next to the BamHI/BglII junction in the pUC19 plasmid. In addition to deletion of the indicated Ha hsp17.7 G4 sequences, the manipulations described above also resulted in minor sequence changes at the G4::GUS junction. The Ha hsp18.6 G2 fusion contained the sHSP genomic sequences in a 2.3-kb XbaI fragment (between positions of approximately 2,170 and +121 [Coca et al., 1996]). This included the promoter, 5'-UTR, and the first 21 amino acids fused to GUS at the XbaI site of pBI 101.2. Maps indicating all sequence differences between the WT and BglII constructs are provided in "Results" (Fig. 1). Details of the chimeric GUS proteins are given in Figure 8.

We produced different primary (T0) transgenic plants using published procedures for tobacco transformation and for the characterization of transgene integration and inheritance (Coca et al., 1996). The progeny (T1 plants) of these transgenic plants were used for the heat shock-induced GUS reporter assays.

Plant Material, RNA Protection, and GUS Reporter Assays

We used total RNA prepared from transgenic plant material: seeds (at different developmental stages) or from seedlings (at 15 d post imbibition) grown either under controlled conditions or heat stressed for 2 h and 30 min at 42°C. Conditions for plant growth and stress treatments have been described before in detail (Coca et al., 1996). Preparation of total RNA samples was as described for sunflower (Helianthus annuus) plant material (Coca et al., 1996). Preparation of riboprobes, RNA hybridization, digestion with RNase A, and subsequent gel electrophoresis analyses were performed essentially as described (Coca et al., 1996). We used per hybridization 10 µg of RNA and 200,000 cpm of each probe. Hybridizations were for 16 h at 48°C. RNase A digestions were for 60 min at 30°C. Digestion products were analyzed in 4% (w/v) acrylamide-urea gels. The NptII probe, used in the protection experiments as internal standard for transgene expression, was prepared as follows. We cloned, in pBluescript SK, an internal DNA fragment from the NptII gene in pBI 101: from PstI (+177 from the ATG) to SphI (+534). The resulting plasmid was linearized with EcoRI and in vitro transcribed using T3 RNA polymerase. We, thus, obtained a 430-nucleotide antisense riboprobe, which should detect protected RNA fragments of 357 nucleotides after hybridization and digestion with RNase A. The Ha hsp17.7 G4::GUS probe was obtained from another pBluescript SK plasmid in which we cloned the sequences present in the WT fusion (Coca et al., 1996) between a HindIII site in Ha hsp17.7 G4 (+93 from transcription initiation) and the unique SnaBI site in the GUS protein-coding sequence. This plasmid was linearized with XhoI and in vitro transcribed using T3 RNA polymerase, resulting in an antisense riboprobe of 554 nucleotides. Hybridizations with this probe should detect protected fragments with different sizes that originate from transcripts of the different chimeric genes: 476 nucleotides in the case of WT and 392 for BglII. Band intensities were quantified by densitometric analyses of digital PICT files. These files were acquired with 600-dots per inch resolution from the original autoradiographs using a UMAX Scanner and Adobe Photoshop 3.0 (Adobe Systems, Mountain View, CA). Densitometry was performed using the Kodak EDAS 2.0 software (Eastman Kodak, Rochester, NY).

The fluorometric GUS assays were performed as previously reported (Coca et al., 1996; Almoguera et al., 1998; Carranco et al., 1999) using plant material from the same experiments described for the RNA analyses. We also investigated potential effects of temperature on the stability (or activity) of the different chimeric proteins encoded by the transgenes. Samples containing these proteins were fluorometrically assayed in the usual conditions and after pretreatment of the reaction mixtures (without the substrate) for different times and at different temperatures. Reactions were started with the addition of the substrate and GUS activity measured as reported (Coca et al., 1996).

Distribution of Materials

All constructs and seeds from plant material described here will be available for academic research by obtaining permission from the authors.

Immunoblotting Detection and Immunoprecipitation of GUS Proteins

Total protein extracts were prepared from frozen plant material, separated in 8% (w/v) SDS-PAGE, and transferred to Immobilon-P membranes (Millipore, Bedford, MA) as described by Almoguera et al. (1993). GUS proteins were detected using as primary antibody the anti-GUS rabbit IgG (H+L) fraction (Molecular Probes, Eugene, OR), at 1:2,500 dilution. We also used as the secondary antibody anti-rabbit IgG horseradish peroxidase conjugate (Promega, Madison, WI) diluted 1:15,000 and the SuperSignal West Pico system (Pierce, Indianapolis). Before immunodetection, filters were stained with 0.2% (w/v) Ponceau-S and photographed.

We used the same antibody for immunoprecipitation of the WT and BglII GUS proteins. Extracts were prepared in non-denaturing conditions (radioimmunoprecipitation assay buffer) and soluble proteins immunoprecipitated by following the procedures described by Krishna et al. (1997). Precipitated protein was loaded in 12% (w/v) SDS-PAGE gels that were stained with a silver staining kit (Bio-Rad, Hercules, CA).