Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

A Plant Small Heat Shock Protein Gene Expressed during Zygotic Embryogenesis but Noninducible by Heat Stress

1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês

10.1074/jbc.272.43.27470

ISSN

1083-351X

Autores

Raúl Carranco, Concepción Almoguera, Juan Jordano,

Tópico(s)

Physiological and biochemical adaptations

Resumo

A small heat shock protein (sHSP) gene from sunflower, Ha hsp17.6 G1, showed expression patterns that differ from what is known for members of this gene family. The mRNAs of this gene accumulated in seeds during late desiccation stages of zygotic embryogenesis but not in response to heat shock in vegetative tissues. The failure to respond to heat shock was independent of the developmental stage after germination and shock temperature. Nuclear run-on analyses demonstrated that transcription from the Ha hsp17.6 G1 promoter is not induced by heat shock. This agrees with the presence, in this promoter, of sequences with little similarity to heat shock elements. Our results show an evolutionary divergence, in the regulation of plant sHSP genes, which has originated stress-responsive genes and nonresponsive members within this gene family. We discuss implications for mechanisms controlling the developmental regulation of sHSP genes in plants. A small heat shock protein (sHSP) gene from sunflower, Ha hsp17.6 G1, showed expression patterns that differ from what is known for members of this gene family. The mRNAs of this gene accumulated in seeds during late desiccation stages of zygotic embryogenesis but not in response to heat shock in vegetative tissues. The failure to respond to heat shock was independent of the developmental stage after germination and shock temperature. Nuclear run-on analyses demonstrated that transcription from the Ha hsp17.6 G1 promoter is not induced by heat shock. This agrees with the presence, in this promoter, of sequences with little similarity to heat shock elements. Our results show an evolutionary divergence, in the regulation of plant sHSP genes, which has originated stress-responsive genes and nonresponsive members within this gene family. We discuss implications for mechanisms controlling the developmental regulation of sHSP genes in plants. One of the characteristics of the plant heat shock response is the synthesis of a large number of different, but evolutionarily related, polypeptides of 17–30 kDa (the sHSPs). 1The abbreviations used are: sHSP(s), small heat shock protein(s); HSE, heat shock element; HSF, heat shock factor; hHSF1, human heat shock factor 1; bp, base pair(s); dpi, days postimbibition; dpa, days postanthesis. 1The abbreviations used are: sHSP(s), small heat shock protein(s); HSE, heat shock element; HSF, heat shock factor; hHSF1, human heat shock factor 1; bp, base pair(s); dpi, days postimbibition; dpa, days postanthesis. In contrast, animals express only one to four sHSPs upon heat shock. The diversification in plants of heat-inducible sHSP genes could be a consequence of sesility; because plants cannot move away from heat, they would have evolved a battery of specialized 舠stress genes,舡 the sHSPs. These are expressed in response to heat in all subcellular compartments and could allow plants to cope better with the stress conditions on site (for review, see Ref. 1Waters E.R. Lee G.J. Vierling E. J. Exp. Bot. 1996; 47: 325-338Crossref Scopus (575) Google Scholar). In animal and plant systems, heat shock genes encoding proteins of higher molecular weight, for example the HSP70s, have been shown to contain heat-inducible and noninducible members (2DeRocher A. Vierling E. Plant Mol. Biol. 1995; 27: 441-456Crossref PubMed Scopus (64) Google Scholar,3Gurley W.B. Key J.L. Biochemistry. 1991; 30: 1-12Crossref PubMed Scopus (68) Google Scholar). In the case of plant sHSPs the evidence for the existence of genes that are not induced by heat shock is weak and indirect, as it is based on the detection in seeds of sHSP isoforms that are different from the heat shock-induced polypeptides (4Coca M.A. Almoguera C. Jordano J. Plant Mol. Biol. 1994; 25: 479-492Crossref PubMed Scopus (124) Google Scholar, 5Zur Nieden U. Neumann D. Bucka A. Nover L. Planta. 1995; 196: 530-538Crossref Scopus (42) Google Scholar). In addition to being part of the heat shock response, some plant sHSP genes have been shown to be expressed at normal growth temperatures during zygotic embryogenesis (4Coca M.A. Almoguera C. Jordano J. Plant Mol. Biol. 1994; 25: 479-492Crossref PubMed Scopus (124) Google Scholar, 5Zur Nieden U. Neumann D. Bucka A. Nover L. Planta. 1995; 196: 530-538Crossref Scopus (42) Google Scholar, 6Almoguera C. Jordano J. Plant Mol. Biol. 1992; 19: 781-792Crossref PubMed Scopus (132) Google Scholar, 7DeRocher A.E. Vierling E. Plant J. 1994; 5: 93-102Crossref Scopus (107) Google Scholar). Developmental regulation studies of plant sHSP genes are scarce. So far, only two plant sHSP promoters and 5′-flanking sequences have been reported to confer regulation to chimeric genes in maturing seeds: those from soybean Gm hsp17.3B (8Prändl R. Kloske E. Schöffl F. Plant Mol. Biol. 1995; 28: 73-82Crossref PubMed Scopus (55) Google Scholar) and sunflower Ha hsp17.7 G4 (9Coca M.A. Almoguera C. Thomas T.L. Jordano J. Plant Mol. Biol. 1996; 31: 863-876Crossref PubMed Scopus (85) Google Scholar). Initial studies have pointed to common control elements between the heat shock response and activation during embryogenesis. For example, the functional implication of HSEs in both processes is supported by results of deletion analysis (8Prändl R. Kloske E. Schöffl F. Plant Mol. Biol. 1995; 28: 73-82Crossref PubMed Scopus (55) Google Scholar, 9Coca M.A. Almoguera C. Thomas T.L. Jordano J. Plant Mol. Biol. 1996; 31: 863-876Crossref PubMed Scopus (85) Google Scholar). Other observations point to the involvement in embryos of distinct control elements, for example, the effect of abi3 mutations on sHSP accumulation inArabidopsis seeds (10Wehmeyer N. Hernandez L.D. Finkelstein R.R. Vierling E. Plant Physiol. 1996; 112: 747-757Crossref PubMed Scopus (210) Google Scholar). It is also clear that not all plant sHSP genes are developmentally regulated during embryogenesis, at least for the most systematically analyzed sunflower (9Coca M.A. Almoguera C. Thomas T.L. Jordano J. Plant Mol. Biol. 1996; 31: 863-876Crossref PubMed Scopus (85) Google Scholar) andArabidopsis genes (10Wehmeyer N. Hernandez L.D. Finkelstein R.R. Vierling E. Plant Physiol. 1996; 112: 747-757Crossref PubMed Scopus (210) Google Scholar). Thus, there is no obvious explanation for the differential regulation of a subset of sHSP genes during embryogenesis, and such regulation might involve control elements that are common and/or distinct from those involved in the heat shock response. In this work, we describe and analyze the genomic sequences of Ha hsp17.6 G1, a sunflower sHSP gene expressed during zygotic embryogenesis (6Almoguera C. Jordano J. Plant Mol. Biol. 1992; 19: 781-792Crossref PubMed Scopus (132) Google Scholar). By detailed analyses of gene-specific mRNA accumulation and transcription, we demonstrate that transcription from the Ha hsp17.6 G1 promoter is not induced in response to heat shock. Interestingly, such a unique expression pattern has been described in other animal genes that are structurally related (11Caspers G.J. Leunissen J.A.M. de Jong W.W. Mol. Biol. Evol. 1995; 40: 238-248Crossref Scopus (308) Google Scholar) to the sHSPs, as for the eye lens-specific αA-crystallins (12Overbeek P.A. Chepelinsky A.B. Khillan J.S. Piatigorsky J. Westphal H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7815-7819Crossref PubMed Scopus (150) Google Scholar) and, more recently, for some αB-crystallins (13Wistow G. Graham C. Biochim. Biophys. Acta. 1995; 1263: 105-113Crossref PubMed Scopus (11) Google Scholar). Thus, our observation provides strong evidence for a similar evolution of the regulation of a member of the sHSP superfamily in the plant kingdom. In addition, the structural and functional characteristics of the Ha hsp17.6 G1 promoter would support developmental control mechanisms, during plant embryogenesis, which are different from those involved in the heat shock response. Two restriction fragments containing the HSE sequences of Ha hsp17.7 G4 and Ha hsp17.6 G1 were purified from 1.67 agarose gels and end labeled with Klenow and dATP33 (14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). TheHa hsp17.6 G1 probe is a 175-nucleotide fragment between theHindIII sites at −126 and +50, and the Ha hsp17.7 G4 probe is a 287-nucleotide fragment that contains promoter sequences between −188 (EcoRV) and +80 (9Coca M.A. Almoguera C. Thomas T.L. Jordano J. Plant Mol. Biol. 1996; 31: 863-876Crossref PubMed Scopus (85) Google Scholar). This fragment was labeled at an EcoRI site from vector polylinker sequences. Binding reactions were performed for 15 min, at 20 °C, in 10 mm Hepes, pH 7.9; 1.5 mm MgCl2; 0.05 mm EDTA; 120 mm NaCl; and 67 glycerol. Reactions included 1 ng of each labeled fragment, 1.8 ॖg of poly(dI-dC) (Pharmacia Biotech Inc.) and 2 ॖg of a protein extract, obtained from Escherichia coli BL21 cells expressing human HSF1 from plasmid pHu HSFM1 (15Rabindran S.K. Giorgi G. Clos J. Wu C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6906-6910Crossref PubMed Scopus (376) Google Scholar). For competition experiments the binding reactions also included a 50-fold molar excess of the unlabeled fragments or of a synthetic, double-stranded, HSE oligonucleotide described by Hübel and Schöffl (16Hübel A. Schöffl F. Plant Mol. Biol. 1994; 26: 353-362Crossref PubMed Scopus (92) Google Scholar). The same molar excess of the 445-bp PvuII fragment from pBluescript SK+ was used as negative control. Subsequent to binding of protein extracts, samples with labeled DNA were subjected to gel shift assays (17Zimarino V. Wu C. Nature. 1987; 327: 727-730Crossref PubMed Scopus (249) Google Scholar). Conditions for growth and heat shock treatments of whole sunflower plants and seedlings (Helianthus annuus L., cv Sunweed, Rhône Poulenc) have been described elsewhere in detail (4Coca M.A. Almoguera C. Jordano J. Plant Mol. Biol. 1994; 25: 479-492Crossref PubMed Scopus (124) Google Scholar, 6Almoguera C. Jordano J. Plant Mol. Biol. 1992; 19: 781-792Crossref PubMed Scopus (132) Google Scholar, 18Almoguera C. Coca M.A. Jordano J. Plant J. 1993; 4: 947-958Crossref Scopus (62) Google Scholar). Embryos were collected at different stages of zygotic embryogenesis under controlled growth conditions and analyzed as reported (6Almoguera C. Jordano J. Plant Mol. Biol. 1992; 19: 781-792Crossref PubMed Scopus (132) Google Scholar). Four days postimbibition (dpi) seedlings were treated with 100 ॖm abscisic acid for 24 h (6Almoguera C. Jordano J. Plant Mol. Biol. 1992; 19: 781-792Crossref PubMed Scopus (132) Google Scholar). RNase A protection assays were performed as described (19Almoguera C. Coca M.A. Jordano J. Plant Physiol. 1995; 107: 765-773Crossref PubMed Scopus (32) Google Scholar). The RNA probes (riboprobes) were prepared by in vitrotranscription with T3 RNA polymerase (19Almoguera C. Coca M.A. Jordano J. Plant Physiol. 1995; 107: 765-773Crossref PubMed Scopus (32) Google Scholar). In the case of Ha hsp17.6 G1, the 813 nucleotide riboprobe contains, in addition to 55 nucleotides from the vector (from the T3 promoter to SmaI in the SK+ polylinker), the noncoding strand sequences of Ha hsp17.6 G1 between −533 and +225. In the case of Ha hsp17.7 G4, we used a 651-nucleotide riboprobe described before (9Coca M.A. Almoguera C. Thomas T.L. Jordano J. Plant Mol. Biol. 1996; 31: 863-876Crossref PubMed Scopus (85) Google Scholar). Intact nuclei were extracted from samples of control (at 14 dpi) and heat-stressed seedlings (40 min at 40 °C) as well as from zygotic embryos (at 20–24 days postanthesis (dpa)) dissected from plants grown under control conditions. The nuclear purification protocol was a modification of one of the procedures (method II) described by Luthe and Quatrano (20Luthe D.S. Quatrano R.S. Plant Physiol. 1980; 65: 305-308Crossref PubMed Google Scholar). After centrifugation through discontinuous Percoll gradient layers, the band of intact nuclei (in the 807 layer) was removed and was washed twice in Honda buffer by centrifugation at 2,500 × g for 8 min at 4 °C. The purified nuclear pellet was resuspended in 5 ml of nuclear resuspension buffer and centrifuged again at 4,000 ×g for 8 min at 4 °C. Nuclei were finally resuspended in 500 ॖl of nuclear resuspension buffer and stored at −80 °C until use. The amount of nuclear DNA in the preparation was quantified following the procedure of Wanner and Gruissem (21Wanner L.A. Gruissem W. Plant Cell. 1991; 3: 1289-1303Crossref PubMed Scopus (102) Google Scholar). Standard transcription assays, containing 100 ॖg of nuclear DNA in a total volume of 100 ॖl, were performed for 20 min at 25 °C. The reactions contained 50 mm Tris-HCl, pH 8.0; 10 mm MgCl2; 75 mm NH4Cl; 0.45 mm KCl; 1 mm MnCl2; 1 mm dithiothreitol; 150 units of RNAsin (Pharmacia); 0.5 mm each ATP, CTP, GTP; 75 ॖCi of [α-32P]UTP (3,000 Ci/mmol); and 127 glycerol. Reactions were stopped and the in vitro transcribed RNA purified as described by Mittler and Zilinskas (22Mittler R. Zilinskas A. Plant J. 1994; 5: 397-405Crossref PubMed Scopus (357) Google Scholar). Typical incorporation in RNA was 1–1.5 × 105 cpm/ॖg of nuclear DNA. Similar amounts of radiolabeled transcripts (≈107 cpm) were used for hybridization analyses. Hybridizations with labeled run-on RNAs were carried out, in DNA excess, with identical Southern blot filters containing ≈0.8 ॖg of different DNA fragments. These fragments were shown to give sHSP-specific hybridization patterns in sunflower (see 舠Results舡). They include the Ha hsp17.6 G1 sequences betweenDdeI (+442) and XbaI (+699); the Ha hsp18.6 G2 sequences between HincII (+486) and ClaI (≈ +762); and the Ha hsp17.7 G4 sequences betweenSacI (+399) and HindIII (+683). Numbers refer to positions with respect to transcription initiation sites. The filters also included 0.6 ॖg of a 950-bp tetraubiquitin DNA fragment (UbiS). This fragment contains cDNA sequences between positions 127 and 1069 (19Almoguera C. Coca M.A. Jordano J. Plant Physiol. 1995; 107: 765-773Crossref PubMed Scopus (32) Google Scholar). Hybridizations were performed at 45 °C for 50 h in 1.3 ml of hybridization buffer. After prehybridization, hybridization, and washing under the described conditions (4Coca M.A. Almoguera C. Jordano J. Plant Mol. Biol. 1994; 25: 479-492Crossref PubMed Scopus (124) Google Scholar, 6Almoguera C. Jordano J. Plant Mol. Biol. 1992; 19: 781-792Crossref PubMed Scopus (132) Google Scholar), filters were exposed for autoradiography. The genomic library described in Coca et al. (9Coca M.A. Almoguera C. Thomas T.L. Jordano J. Plant Mol. Biol. 1996; 31: 863-876Crossref PubMed Scopus (85) Google Scholar) was rescreened using as a probe the complete cDNA Ha hsp17.6 (6Almoguera C. Jordano J. Plant Mol. Biol. 1992; 19: 781-792Crossref PubMed Scopus (132) Google Scholar). A genomic clone that corresponded exactly to the transcribed sequences in the original cDNA was isolated and accordingly named Ha hsp17.6 G1. This gene encodes HSP17.6, a canonical class I sHSP that, as well as the corresponding mRNA, accumulates during zygotic embryogenesis in the absence of exogenous stress (4Coca M.A. Almoguera C. Jordano J. Plant Mol. Biol. 1994; 25: 479-492Crossref PubMed Scopus (124) Google Scholar, 6Almoguera C. Jordano J. Plant Mol. Biol. 1992; 19: 781-792Crossref PubMed Scopus (132) Google Scholar). A 4.5-kilobase SacI fragment that contained the coding region was subcloned in pBluescript SK+, and the nucleotide sequence of this region, as well as 1521 bp of 5′-flanking and 723 bp of 3′-flanking sequence, was determined on both strands of DNA (Fig. 1 A and data not shown). Initiation of transcription in mRNAs from embryos was determined by primer extension using a synthetic primer from +144 to +120 in Ha hsp17.6 G1 (not shown) and confirmed by RNase A protection assays (Fig. 2; other analyses, in higher resolution gels, not shown). These two techniques detected two close transcription start sites at 35 and 43 nucleotides upstream from the initiation codon (indicated by arrows andnumbers in Fig. 1 A). The first of these two sites (site 1) conforms better to the consensus sequences for transcriptional initiation of plant genes (23Joshi C.P. Nucleic Acids Res. 1987; 15: 6643-6653Crossref PubMed Scopus (683) Google Scholar) and is placed at a normal distance from the putative TATA box. Thus, site 1 was chosen to start numbering thehsp17.6 nucleotide sequence (Fig. 1 A).Figure 2Accumulation patterns of Ha hsp17.6 G1 mRNAs during zygotic embryogenesis and seed germination. Different total RNA samples were hybridized toHa hsp17.6 G1 riboprobe. RNA hybrids were digested with RNase A and the products analyzed by 47 polyacrylamide gel electrophoresis. The RNA samples were from yeast t-RNA (t), embryos at different developmental stages (numbers onembryo lanes correspond to age in dpa), and from seedlings (at 4 dpi), either under control conditions (lane 1) or after treatment with abscisic acid (lane 2). In the case of seedlings, the same RNA samples of lanes 1 and 2were also hybridized to the Ha hsp17.7 G4 riboprobe to control induction by abscisic acid (lanes 3 and4). Arrows on the right ofpanels mark the fully protected RNA fragments detected with either riboprobe. Numbers 1 and 2 correspond to the two transcriptional initiation sites of Ha hsp17.6 G1(see Fig. 1 A). Lane m shows DNA size markers, andlane p is undigested Ha hsp17.6 G1 probe.View Large Image Figure ViewerDownload Hi-res image Download (PPT) RNase A protection assays with total RNA samples from staged embryos determined the accumulation patterns of these transcripts during normal zygotic embryogenesis, with messages appearing around 12 dpa and drastically increasing their abundance from 18 dpa, coincident with seed desiccation during late embryogenesis. Transcripts originating from the two transcription initiation sites accumulated to similar levels during late embryogenesis, indicating their functional equivalence (Fig. 2, 18 and 20 dpa). The mRNAs that accumulated during seed maturation disappeared during germination by 4 dpi (Fig. 2, seedlings, lane 1). Exogenous abscisic acid treatments of seedlings at this stage failed to induce the accumulation of the same transcripts (Fig. 2, seedlings; comparelanes 1 and 2); whereas such treatment induced accumulation of mRNAs from the homologous gene Ha hsp17.7 G4 (Fig. 2, seedlings; compare lanes 3 and4). Inspection of the sequences in the proximal promoter region of Ha hsp17.6 G1 revealed unusual characteristics when compared with the same regions of two homologous sHSP genes from sunflower, Ha hsp17.7 G4 and Ha hsp18.6 G2 (9, Fig. 1). Most noteworthy was the absence in the proximal promoter region of clearly defined arrays of potential HSEs as those described previously for Ha hsp17.7 G4, Ha hsp18.6 G2(respectively, Fig. 1 B and C; Ref. 9Coca M.A. Almoguera C. Thomas T.L. Jordano J. Plant Mol. Biol. 1996; 31: 863-876Crossref PubMed Scopus (85) Google Scholar), and for most plant sHSP genes (for review, see Ref. 3Gurley W.B. Key J.L. Biochemistry. 1991; 30: 1-12Crossref PubMed Scopus (68) Google Scholar). These arrays normally comprise alternating nGAAn/nTTCn repeats, in which the G or C nucleotides situated at position 1 or 3 of the core repeats have been shown to be crucial for HSF binding and heat-induced activation (24Dulce Barros M. Czarnecka E. Gurley W.B. Plant Mol. Biol. 1992; 19: 665-675Crossref PubMed Scopus (41) Google Scholar,25Fernandes M. Xiao H. Lis J.T. Nucleic Acids Res. 1994; 22: 167-173Crossref PubMed Scopus (148) Google Scholar). A functional HSE is thought to require at least three of these perfect repeats in a proper promoter context (26Fernandes M. O'Brien T. Lis J.T. Morimoto R.I. Tissières A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 375-393Google Scholar). The nucleotide sequence of Ha hsp17.6 G1 showed only three alternating repeats (Fig. 1 A), of which the one situated in the middle (TTt) does not conform to the consensus sequence for the core repeat at the crucial third position. A fourth imperfect repeat that fits the core consensus at the crucial first position (GAt) is placed upstream of the other three (Fig. 1 A). This is a potentially correct spatial alignment, as functional HSEs can tolerate a 5-bp gap between repeats (27Amin J. Ananthan J. Voellmy R. Mol. Cell Biol. 1988; 8: 3761-3769Crossref PubMed Scopus (433) Google Scholar). The four core repeats that comprise the sole putative HSE array of Ha hsp17.6 G1 are placed at considerable distance (50 bp) upstream from the putative TATA box. This position would correspond better to that of the most 5′-distal of the two HSE arrays normally found in the proximal promoter region of other plant sHSP genes, including the Ha hsp17.7 G4 and Ha hsp18.6 G2 promoters (site II, Fig. 1, B and C; Refs. 3Gurley W.B. Key J.L. Biochemistry. 1991; 30: 1-12Crossref PubMed Scopus (68) Google Scholar and 9Coca M.A. Almoguera C. Thomas T.L. Jordano J. Plant Mol. Biol. 1996; 31: 863-876Crossref PubMed Scopus (85) Google Scholar). To investigate the binding potential of the putative HSE array inHa hsp17.6 G1, we conducted mobility shift assays using hHSF1 (15Rabindran S.K. Giorgi G. Clos J. Wu C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6906-6910Crossref PubMed Scopus (376) Google Scholar) and two radiolabeled DNA fragments containing the HSE arrays of Ha hsp17.7 G4 or Ha hsp17.6 G1. Binding of hHSF1 to each fragment was compared by performing competition experiments using the same molar excesses of unlabeled fragments, including a positive control (the same fragment used as labeled probe) and a negative control (a DNA fragment from plasmid vector sequences). We detected binding of hHSF1 to a fragment containing the imperfect HSE array in Ha hsp17.6 G1 (Fig.3, lane 2). The specificity of binding was demonstrated by competition experiments. Addition of vector DNA fragment did not affect the retarded band, whereas the same excess of either unlabeled Ha hsp17.6 G1 or Ha hsp17.7 G4 fragments affected detection of the complexes. The Ha hsp17.7 G4 fragment was the most efficient competitor (Fig. 3,lanes 3–5). This specificity was verified further by other experiments using various molar excesses of the same and other DNA fragments, including one oligonucleotide with a perfect synthetic HSE array (not shown). We also observed similar complexes using the labeledHa hsp17.7 G4 probe, and these complexes could be competed efficiently by a 50-fold molar excess of the unlabeled Ha hsp17.7 G4 fragment (Fig. 3, lanes 7 and 10); but in contrast, the same excess of the unlabeled Ha hsp17.6 G1fragment was as an inefficient competitor as the vector DNA fragment (Fig. 3, lanes 8 and 9). Thus, reverse competition experiments indicated that even if able to bind hHSF1in vitro, the Ha hsp17.6 G1 sequences had a much lower affinity for this factor than the HSEs of Ha hsp17.7 G4. In conclusion, the promoter region of Ha hsp17.6 G1contains a short and imperfect HSE array compared with other similar sHSP genes. This raised doubts over the heat induction of this promoter. In previous studies, we detected by Northern hybridization the heat-induced accumulation of homologous sHSP mRNAs, using as probe the complete Ha hsp17.6 cDNA (6Almoguera C. Jordano J. Plant Mol. Biol. 1992; 19: 781-792Crossref PubMed Scopus (132) Google Scholar, 18Almoguera C. Coca M.A. Jordano J. Plant J. 1993; 4: 947-958Crossref Scopus (62) Google Scholar). Because this probe is not gene-specific, we decided to investigate the heat stress expression patterns of the Ha hsp17.6 G1 mRNAs using the more sensitive technique of ribonuclease protection, which has been used successfully to investigate gene-specific expression patterns of plant sHSP genes (9Coca M.A. Almoguera C. Thomas T.L. Jordano J. Plant Mol. Biol. 1996; 31: 863-876Crossref PubMed Scopus (85) Google Scholar, 28Krishna P. Felsheim R.F. Larkin J.C. Das A. Plant Physiol. 1992; 100: 1772-1779Crossref PubMed Scopus (22) Google Scholar). Heat-induced accumulation of the Ha hsp17.6 G1 mRNAs was investigated using total RNA samples from seedlings and plants (representative results in Figs. 4 and5). The same RNA samples were hybridized to either Ha hsp17.7 G4 or to Ha hsp17.6 G1riboprobes. Ha hsp17.7 G4 was used as a positive control for heat induction in sunflower (9Coca M.A. Almoguera C. Thomas T.L. Jordano J. Plant Mol. Biol. 1996; 31: 863-876Crossref PubMed Scopus (85) Google Scholar). Messages from the Ha hsp17.6 G1 gene were detected only in a positive control sample from seeds (Fig. 4 A, lane s) but not in control or heat-stressed samples from seedlings and stems or leaves from adult plants (Fig. 4 A, lanes 1–6). Only an almost undetectable signal was barely visible in the heat-stressed stem sample (Fig. 4 A, lane 6). In contrast, the analysis of the same RNA samples with the Ha hsp17.7 G4 probe showed that messages from this gene accumulated in all organs after heat shock treatment (Fig. 4 B, lanes 2, 4, and6), which was evident even after shorter autoradiography exposures than those used for detection of Ha hsp17.6 G1mRNAs (Fig. 4, compare panels A and B).Figure 5Effect of temperature on Ha hsp17.6 G1 mRNA accumulation. Identical RNA samples from seedlings (4 dpi) at control growth temperature (panels C, 20 °C) or after heat shock treatments for 2 h 30 min at different temperatures (lane numbers °C) were analyzed by RNase A protection using the indicated riboprobes. The arrowmarks the position of the fully protected fragment from the Ha hsp17.7 G4 mRNAs. The two asterisks indicate the expected position for the two fragments predicted from full protection of Ha hsp17.6 G1 mRNAs. The undigested riboprobes used for hybridizations are shown: Ha hsp17.6 G1 (p1), and Ha hsp17.7 G4 (p2). The picture corresponds to an autoradiogram after a 22-h exposure. The rest of thesymbols are as in the legend of Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The observed lack of heat shock response of Ha hsp17.6 G1could be dependent on temperature, as not all plant heat shock genes would optimally respond at the same temperature. We investigated this possibility by performing different heat shock treatments at various temperatures within natural growth conditions for sunflower. Heat-induced accumulation of Ha hsp17.6 G1 and Ha hsp17.7 G4 messages was investigated by RNase A protection as described above. The heat-induced accumulation of Ha hsp17.7 G4 messages was observed from 30 °C, reaching maximal levels at 40 °C and decreasing slightly at 45 °C (Fig. 5). The same RNA samples analyzed with the Ha hsp17.6 G1 riboprobe did not show heat shock-induced accumulation of Ha hsp17.6 G1messages in any of the tested stress conditions (Fig. 5). The experiments described above established that the mRNAs of Ha hsp17.6 G1 do not accumulate in response to heat stress, in different organs and developmental stages, after germination of sunflower. Our observation could be explained by a peculiar transcriptional regulation of Ha hsp17.6 G1 in response to heat shock. To investigate this possibility, we carried out run-on transcription analyses with isolated nuclei from embryos at normal growth temperature and from control and heat-stressed seedlings. As a control of the transcriptional activity by RNA polymerase II, we used the detection of transcripts of sunflower polyubiquitin genes expressed in all of these experimental conditions (19Almoguera C. Coca M.A. Jordano J. Plant Physiol. 1995; 107: 765-773Crossref PubMed Scopus (32) Google Scholar). The transcripts of two heat-inducible genes, Ha hsp18.6 G2 and Ha hsp17.7 G4 (9Coca M.A. Almoguera C. Thomas T.L. Jordano J. Plant Mol. Biol. 1996; 31: 863-876Crossref PubMed Scopus (85) Google Scholar), were used as a control for transcriptional activation by heat shock; and those of Ha hsp17.7 G4 served also as a control for transcriptional activation in embryos. The transcripts fromHa hsp17.6 G1, Ha hsp17.7 G4, and Ha hsp18.6 G2 were distinguished by hybridization with gene-specific probes. The specificity of probe hybridization was verified with Southern blot analysis using recombinant phage and sunflower genomic DNA (not shown). Transcriptional run-on analysis confirmed the heat induction of Ha hsp17.7 G4 and Ha hsp18.6 G2(Fig. 6, lanes 3 and4, compare control and heat shock) but failed to detect heat shock-induced transcription from the Ha hsp17.6 G1 promoter (Fig. 6, lane 2, compare control and heat shock). In embryos, however, this analysis detected transcriptional activity ofHa hsp17.6 G1 and Ha hsp17.7 G4 (Fig. 6, embryos,lanes 2 and 4), although it failed to detect significant transcription from the Ha hsp18.6 G2 promoter (Fig. 6, embryos, lane 3). In all cases the ubiquitin probe confirmed the transcriptional activity of RNA polymerase II in the isolated nuclei (Fig. 6, lane 1). The finding of a developmentally regulated plant sHSP gene transcribed during zygotic embryogenesis, but which is not responsive to heat shock in vegetative tissues, demonstrates the evolution of the regulation in members of the sHSP gene superfamily in the plant kingdom. HSP families known to contain genes noninducible by heat shock include only those encoding large proteins, for example, the HSP70s. Some of these genes are developmentally regulated, whereas others show constitutive expression irrespective of development (i.e.Ref. 2DeRocher A. Vierling E. Plant Mol. Biol. 1995; 27: 441-456Crossref PubMed Scopus (64) Google Scholar). An accepted evolutionary scenario for the origin of constitutive HSP genes is gradual divergence from heat-inducible ancestors, as heat-induction is regarded as an ancient and well conserved trait. It is assumed that this divergence involved changes in crucial cis-elements for the heat shock response (i.e. the HSEs). The presence of relictic (low homology) HSEs is a trademark of plant constitutive HSP genes (for review, see Ref. 3Gurley W.B. Key J.L. Biochemistry. 1991; 30: 1-12Crossref PubMed Scopus (68) Google Scholar and references therein). This view fits nicely with the unusual structure and in vitro HSF binding characteristics of the HSE region in Ha hsp17.6 G1 compared with those of other plant sHSP genes, including two homologues from sunflower (Figs. 1 and3). The putative HSE region in Ha hsp17.6 G1 (Fig.1 A) would not support either efficient HSF1 binding in vitro (Fig. 3) or heat shock-induced transcriptional activation (Fig. 6, heat shock panel). The HSEs in Ha hsp17.7 G4 show intermediate characteristics and still support heat shock induction (Figs. 1 B and 6 and Ref. 9Coca M.A. Almoguera C. Thomas T.L. Jordano J. Plant Mol. Biol. 1996; 31: 863-876Crossref PubMed Scopus (85) Google Scholar). This is indicative of less divergence from the features observed in Ha hsp18.6 G2(Fig. 1 C) and in other plant sHSP promoters (for review, see Ref. 3Gurley W.B. Key J.L. Biochemistry. 1991; 30: 1-12Crossref PubMed Scopus (68) Google Scholar). Interestingly, there are precedents for a similar evolution of the regulation of α-crystallin genes in animals. These genes encode proteins (the A and B subunits of α-crystallins) expressed mostly in the eye lens. The α-crystallins are structurally related to the sHSPs and might have evolved from a common ancestor (11Caspers G.J. Leunissen J.A.M. de Jong W.W. Mol. Biol. Evol. 1995; 40: 238-248Crossref Scopus (308) Google Scholar). Whereas most of the αB-crystallin promoters retain HSEs and heat inducibility, many αA-crystallin and some peculiar αB-crystallin promoters do not contain HSEs and are not heat-inducible (12Overbeek P.A. Chepelinsky A.B. Khillan J.S. Piatigorsky J. Westphal H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7815-7819Crossref PubMed Scopus (150) Google Scholar, 13Wistow G. Graham C. Biochim. Biophys. Acta. 1995; 1263: 105-113Crossref PubMed Scopus (11) Google Scholar). Other work has indicated that a sHSP gene from chicken (hsp23) is not transcriptionally activated in response to heat shock in embryo cells (29Edington B.V. Hightower L.E. Mol. Cell Biol. 1990; 10: 4886-4898Crossref PubMed Scopus (26) Google Scholar). Our results show that an apparently parallel evolution has taken place in plants for other members of the sHSP superfamily, raising new questions on the origins of the differentially regulated genes. The observation of efficient transcription from different plant sHSP promoters in seeds, regardless of the presence, or absence, of high homology HSEs puts some constraints to current models explaining the developmental regulation of these genes (9Coca M.A. Almoguera C. Thomas T.L. Jordano J. Plant Mol. Biol. 1996; 31: 863-876Crossref PubMed Scopus (85) Google Scholar, 30Prändl R. Schöffl F. Plant Mol. Biol. 1996; 31: 157-162Crossref PubMed Google Scholar). A general involvement of HSEs and of trans-acting factors similar to mammalian HSF1 does not fit the available data for the sunflower genes. Thus, transcription from the Ha hsp18.6 G2 promoter is not substantially active in zygotic embryos, despite its efficient heat induction in vegetative tissues, consistent with the presence of high homology HSEs at a proximal location (Figs. 1 C and 6). In addition, Ha hsp17.6 G1 and Ha hsp17.7 G4 provide examples of transcriptional activation in embryos (Fig. 6), respectively, without and with recognizable HSEs (Fig. 1, Aand B, and Fig. 3). In the case of Ha hsp17.7 G4, recent mutagenesis analyses of its HSEs determined that heat induction of this gene in vegetative tissues can be eliminated without effects on its developmental regulation during early seed maturation. However, these mutations reduced expression of Ha hsp17.7 G4 during later seed desiccation stages. 2C. Almoguera, P. Prieto-Dapena, and J. Jordano, submitted for publication. In the case ofHa hsp17.6 G1, natural evolution produced much more diverged HSEs than in any of the tested Ha hsp17.7 G4 mutations; and yet Ha hsp17.6 G1, despite not responding to heat shock, is expressed efficiently during late embryogenesis. An involvement of HSEs in the developmental regulation of Ha hsp17.6 G1 during seed desiccation seems unlikely unless these elements would bind HSFs (o even different factors) with sequence specificity different from that of mammalian HSF1. The multiplicity and divergence of plant HSFs, still uncharacterized for their possible DNA binding and functional diversity (for review, see Ref. 31Nover L. Scharf K.D. Gagliardi D. Vergne P. Czarnecka-Verner E. Gurley W.B. Cell Stress Chaperones. 1996; 1: 215-223Crossref PubMed Google Scholar), could allow the specialization of some HSFs in developmental regulation, as observed similarly in animal systems (32Morimoto R.I. Jurivich D.A. Kroeger P.E. Mathur S.K. Murphy S.P. Nakai A. Sarge K. Abravaya K. Sistonen L.T. Morimoto R.I. Tissières A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 417-455Google Scholar). Ha hsp17.6 G1 and Ha hsp17.7 G4 provide unique opportunities to investigate the molecular basis of developmental regulation of plant sHSP genes during zygotic embryogenesis. They constitute so far a unique pair of homologous genes, expressed with similar patterns in the same species, for which sequence information and preliminary characterization are available. Sequence analysis and expression data indicate that their developmental expression in seeds might be conferred by distinct mechanisms. Future work will elucidate these control mechanisms. We are grateful to Dr. Carl Wu (National Institutes of Health) for the gift of plasmid pHu HSFM1. We thank Drs. Josep Casadesus and Eduardo Santero (Department of Genetics, University of Sevilla) for critical reviews of this manuscript.

Referência(s)