RESUMEN
A new member of the GT-2 family of transcription factors, GmGT-2, was isolated from soybean while screening a cDNA library with a protein binding site (D1) in the promoter of Aux28, a member of the Aux/IAA family of auxin-responsive genes. GmGT-2 possesses various primary amino acid sequence characteristics common to all GT-2 factors thus far isolated, including sequence identity in the twin trihelix DNA-binding domains. Recombinant GmGT-2 expressed in Escherichia coli binds oligotetramers of both D1 and various GT-boxes. However, unlike other known members of the GT-2 family, GmGT-2 message levels are down-regulated by light in a phytochrome-dependent manner. Evidence is presented that the expression levels of Aux28 mRNA are also down-regulated by phytochrome. These results and other referenced data implicate the possible convergence of phytochrome and auxin signaling pathways.
Asunto(s)
Glycine max/genética , Luz , Fitocromo/fisiología , Factores de Transcripción/genética , Secuencia de Aminoácidos , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Plantas/química , ADN de Plantas/genética , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Hipocótilo/genética , Hipocótilo/crecimiento & desarrollo , Hipocótilo/metabolismo , Ácidos Indolacéticos/farmacología , Datos de Secuencia Molecular , Proteínas de Plantas/efectos de los fármacos , Proteínas de Plantas/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Glycine max/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
Two separate nuclear binding activities (B1 and B2) in the soybean apical hypocotyl have been identified that interact with a palindromic C-box sequence (TGACGTCA) and which are developmentally regulated in an inverse manner. The bZIP factors responsible for these two binding activities, B1 and B2, were isolated from a cDNA library and designated STGA1 and STFs (STF1 and STF2), respectively. Sequence analysis shows that the STFs contain both a zinc-finger domain and a bZIP domain. The two zinc finger sequences of Cys4-Cys4 are most related to the RING zinc-finger motif carrying a Cys3-His-Cys4. In addition the bZIP domain of STFs is highly homologous to the HY5 protein of Arabidopsis. DNA binding studies revealed that STF1 binding to the TGACGT sequence requires distinct flanking sequences. Furthermore, STF1 binds to the Hex sequence as a heterodimer with G-box binding factors (GBFs), a feature not observed with STGA1. Since STF1 expression is most prevalent in apical and elongating hypocotyls, it is proposed that STF1 may be a transcription factor involved in the process of hypocotyl elongation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Glycine max/metabolismo , Proteínas de Soja , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Sitios de Unión , ADN Complementario , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Factores de Unión a la G-Box , Biblioteca de Genes , Leucina Zippers , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Dedos de ZincRESUMEN
The effect of the proline analog azetidine-2-carboxylic acid (Aze) on the induction and the regulation of heat-shock (HS) mRNA accumulation and heat-shock protein (HSP) synthesis in soybean (Glycine max) seedlings was studied. Treatment with Aze elicited an HS-like response at the normal growth temperature, 28[deg]C, with seven of nine HS cDNA clones tested. Two cDNA clones, Gm-Hsp22.5 and pFS2033, share 78% identity; however, transcripts hybridizing to GmHsp22.5 but not pFS2033 accumulated with Aze treatment at 28[deg]C. Substantial incorporation of radioactive amino acid into high molecular weight HSPs but not low molecular weight HSPs was observed in vivo during Aze treatment at 28[deg]C. Low molecular weight HSPs were detected using antibodies raised against an abundant member of low molecular weight class I HSPs, indicating that low molecular weight HSPs were synthesized at normal growth temperatures during Aze treatment despite a lack of substantial in vivo radioactive amino acid incorporation. In summary, Aze treatment induced accumulation of most but not all HS mRNAs and HSPs in soybean seedlings; the observations presented here suggest differential regulation among various HS genes at the transcriptional and posttranscriptional levels.
RESUMEN
Three cDNA clones (GmHSP23.9, GmHSP22.3, and GmHSP22.5) representing three different members of the low-molecular-weight (LMW) heat shock protein (HSP) gene superfamily were isolated and characterized. A fourth cDNA clone, pFS2033, was partially characterized previously as a full-length genomic clone GmHSP22.0. The deduced amino acid sequences of all four cDNA clones have the conserved carboxyl-terminal LMW HSP domain. Sequence and hydropathy analyses of GmHSP22, GmHSP22.3, and GmHSP22.5, representing HSPs in the 20 to 24 kDa range, indicate they contain amino-terminal signal peptides. The mRNAs from GmHSP22, GmHSP22.3, and GmHSP22.5 were preferentially associated in vivo with endoplasmic reticulum (ER)-bound polysomes. GmHSP22 and GmHSP22.5 encode strikingly similar proteins; they are 78% identical and 90% conserved at the amino acid sequence level, and both possess the C-terminal tetrapeptide KDEL which is similar to the consensus ER retention motif KDEL; the encoded polypeptides can be clearly resolved from each other by two-dimensional gel analysis of their hybrid-arrest translation products. GmHSP22.3 is less closely related to GmHSP22 (48% identical and 70% conserved) and GmHSP22.5 (47% identical and 65% conserved). The fourth cDNA clone, GmHSP23.9, encodes a HSP of ca. 24 kDa with an amino terminus that has characteristics of some mitochondrial transit sequences, and in contrast to GmHSP22, GmHSP22.3, and GmHSP22.5, the corresponding mRNA is preferentially associated in vivo with free polysomes. It is proposed that the LMW HSP gene superfamily be expanded to at least six classes to include a mitochondrial class and an additional endomembrane class of LMW HSPs.
Asunto(s)
Glycine max/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Biblioteca de Genes , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/clasificación , Membranas/química , Datos de Secuencia Molecular , Polirribosomas/química , Conformación Proteica , ARN de Planta/análisis , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
G-box binding factors (GBFs) constitute a family of plant DNA-binding proteins that bind to the G-box motif, a regulatory cis element present in many plant genes with a palindromic DNA motif of CACGTG. Previously TCCACGTGTC, a G-box motif, from an auxin responsive gene GmAux28 has been identified as a sequence-specific protein-binding site. Here the isolation of two soybean cDNA clones, referred to as SGBF-1 and SGBF-2, encoding proteins which bind to the G-box motif is reported. The primary structure of SGBF-1 and SGBF-2 predicts that these proteins contain a basic leucine zipper (bZIP) DNA-binding domain and an N-terminal proline-rich domain. A dramatic difference in the pattern of protein-DNA complex formation was observed when recombinant SGBF-1 and SGBF-2 proteins were analyzed by electrophoretic mobility shift assays (EMSAs). The SGBF-1 binding pattern obtained with the G-box probe resulted in three major retarded bands while the SGBF-2 formed a single complex. This shows that the characteristically diffuse banding pattern of plant nuclear proteins interacting with the G-box is also observed in a binding assay using only one recombinant GBF. EMSAs were performed with a few selected binding sequences to study the effect of flanking nucleotides to the hexanucleotide G-box core motif. The binding specificity of the SGBF proteins resembles that described for type A cauliflower nuclear G-box binding proteins which bind class I G-box elements [(G/T)(C/A)CACGTG(G/T)(A/C)]. Phylogenetic analysis of 13 GBF-like proteins from various plant species reveals that the SGBF-1 and SGBF-2 proteins belong to different lineages, suggesting that they may have distinct functions in activating transcription.
Asunto(s)
ADN de Plantas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Glycine max/genética , Ácidos Indolacéticos/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción , Secuencia de Aminoácidos , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Northern Blotting , Southern Blotting , Clonación Molecular , ADN Complementario/genética , ADN de Plantas/genética , Proteínas de Unión al ADN/genética , Factores de Unión a la G-Box , Genes de Plantas/genética , Datos de Secuencia Molecular , Familia de Multigenes/genética , Unión Proteica , Conformación Proteica , Análisis de Secuencia de ADNRESUMEN
A cDNA clone encoding a 101-kD heat shock protein (HSP101) of soybean was isolated and sequenced. Genomic DNA gel blot analysis indicated that the corresponding gene is a member of a multigene family. The mRNA for HSP101 was not detected in 2-day-old etiolated soybean seedlings grown at 28 degrees C but was induced by elevated temperatures. DNA sequence comparison has shown that the corresponding gene belongs to the Clp (caseinolytic protease) (or Hsp100) gene family, which is evolutionarily conserved and found in both prokaryotes and eukaryotes. On the basis of the spacer length between the two conserved ATP binding regions, this gene has been identified as a member of the ClpB subfamily. Unlike other Clp genes previously isolated from higher plants, the expression of this soybean Hsp101 gene is heat inducible, and it does not have an N-terminal signal peptide for targeting to chloroplasts. Transformation of the soybean Hsp101 gene into a yeast HSP104 deletion mutant complemented restoration of acquired thermotolerance, a process in which cells survive an otherwise lethal heat stress after they are given a permissive heat treatment.
Asunto(s)
Adaptación Fisiológica/genética , Prueba de Complementación Genética , Glycine max/genética , Proteínas de Choque Térmico/genética , Proteínas de Plantas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario , ADN de Plantas , Proteínas de Choque Térmico/fisiología , Calor , Datos de Secuencia Molecular , Mutación , Eliminación de SecuenciaRESUMEN
We have characterized three different soybean (Glycine max) mRNAs that encode apoproteins of extensins, a family of cell wall hydroxyproline-rich glycoproteins (HRGPs). These transcripts encoded distinctive Tyr-rich proteins containing characteristic Ser-Pro4 sequences organized in higher-order repetitive units. The first transcript encoded an extensin SbHRGP-1 containing the 16-amino acid repeat Ser-Pro4-Ser-Pro-Ser-Pro4-Tyr-Val-Tyr-Lys, with Val occasionally replaced by Ile or Tyr. The second transcript encoded the SbHRGP-2 protein containing the 16-amino acid repeat Ser-Pro4-Ser-Pro-Ser-Pro4-Tyr-Tyr-Tyr-Lys/His. The third transcript encoded the SbHRGP-3 protein containing a variant of 9- or 10-amino acid canonical repeats: Ser-Pro4-Tyr-Lys-Tyr-Pro, Ser-Pro5-Tyr-Lys-Tyr-Pro, and Ser-Pro4-Val-Tyr-Lys-Tyr-Lys, respectively. The dramatic amino acid substitutions in the Tyr-rich blocks (Tyr-X-Tyr-Lys) among these HRGPs indicate that each SbHRGP may have a different function in cell wall architecture.
Asunto(s)
Glycine max/genética , Glicoproteínas/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Five constructions containing deletions of the promoter from an auxin-inducible gene of Arabidopsis thaliana, AtAux2-11, were fused to the coding region of the reporter gene LacZ, which encodes beta-galactosidase, and a polyadenylation 3'-untranslated nopaline synthase sequence from Agrobacterium. These chimeric genes were introduced into Arabidopsis by Agrobacterium tumefaciens-mediated transformation, and expression of the gene was examined by spectrophotometric and histochemical analyses. A 600 bp fragment from the AtAux2-11 promoter conferred histochemical patterns of staining similar to the longest 5' promoter tested, a 3.0 kb fragment. Localization of AtAux2-11/LacZ activity in the transgenic plants revealed spatial and temporal expression patterns that correlated with tissues and cells undergoing physiological processes modulated by auxin. LacZ activity was expressed in the elongating region of roots, etiolated hypocotyls, and anther filaments. Expression was detected in the vascular cylinder of the root and the vascular tissue, epidermis, and cortex of the hypocotyl, and filament. The AtAux2-11/LacZ gene was preferentially expressed in cells on the elongating side of hypocotyls undergoing gravitropic curvature. Expression of the chimeric gene in the hypocotyls of light-grown seedlings was less than that in etiolated seedling hypocotyls. The AtAux2-11/LacZ gene was active in the root cap, and expression in the root stele increased at sites of lateral root initiation. Staining was evident in cell types that develop lignified cell walls, e.g. trichomes, anther endothecial cells, and especially developing xylem. The chimeric gene was not expressed in primary meristems. While the magnitude of expression increased after application of exogenous auxin (2,4-D), the histochemical localization of AtAux2-11/LacZ remained unchanged. Transgenic plants with a 600 bp promoter construct (-0.6 kb AtAux2-11/LacZ) had higher levels of basal and auxin-inducible expression than plants with a 3.0 kb promoter construct. Transgenic plants with a -500 bp promoter had levels of expression similar to the -3.0 kb construct. The -0.6 kb AtAux2-11/LacZ gene responded maximally to a concentration of 5 x 10(-6) to 5 x 10(-5) M 2,4-D and was responsive to as little as 5 x 10(-8) M. The evidence presented here suggests that this gene may play a role in several auxin-mediated developmental and physiological processes.
Asunto(s)
Arabidopsis/genética , Genes de Plantas , Ácidos Indolacéticos/fisiología , Aminoácido Oxidorreductasas/biosíntesis , Aminoácido Oxidorreductasas/genética , Arabidopsis/anatomía & histología , Arabidopsis/crecimiento & desarrollo , Secuencia de Bases , ADN , Regulación de la Expresión Génica , Operón Lac , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Rhizobium/enzimología , Rhizobium/genética , Semillas/metabolismo , Transformación Genética , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
The effects of expressing a chimeric gene consisting of a soybean heat shock gene promoter and a sequence that encodes an enzyme catalyzing the synthesis of a potent phytohormone, the cytokinin iPMP, have been analyzed in transgenic tobacco plants. The production of cytokinin endogenously produced several effects previously undocumented. The differentiation of shoots independent of exogenous cytokinin from heat-treated transgenic plant leaf explants demonstrates that long-term heat treatments do not interfere with complex developmental processes. This extends the potential usefulness of heat shock gene promoters to conditionally express genes during windows of development that span several weeks.
Asunto(s)
Transferasas Alquil y Aril , Citocininas/biosíntesis , Regulación de la Expresión Génica , Glycine max/genética , Nicotiana/metabolismo , Plantas Tóxicas , Adenosina/análogos & derivados , Adenosina/análisis , Citocininas/farmacología , Inducción Enzimática , Genes de Plantas/genética , Proteínas de Choque Térmico/genética , Calor , Isopenteniladenosina/análogos & derivados , Isopenteniladenosina/análisis , Morfogénesis/efectos de los fármacos , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Nicotiana/efectos de los fármacos , Nicotiana/genética , Transferasas/genética , Transferasas/metabolismoRESUMEN
Five cDNA clones (ADR6, ADR11-1, ADR11-2, ADR12-1 and ADR12-2), representing three families of auxin down-regulated (ADR) genes were isolated and characterized. These were isolated by screening a lambda Zap cDNA library with the partial cDNA clones p6, p11 and p12, isolated earlier (Baulcombe and Key, J Biol Chem 255: 8907-8913, 1980). Hybrid-select translation of ADR6, ADR11-2 and ADR12-2 clones produced polypeptides of 33 kDa 22.5 kDa and a 6 and 7 kDa respectively, when analyzed by SDS-PAGE. ADR6 and ADR12-2 gave one and two spots, respectively, on an IEF-SDS 2D gel. ADR11-2 probably encodes a basic protein as it was only resolved on non-equilibrium pH gradient gel electrophoresis (NEPHGE). Genomic Southern blot analysis of ADR6, ADR11 and ADR12 suggests that each represents a small multigene family. The RNA levels corresponding to ADR6, ADR11 and ADR12 decrease in response to applied auxin by 100-, 15- and 10-fold, respectively (Baulcombe and Key, 1980). Runoff transcription, done in the presence and absence of auxin, showed that the rate of transcription of the genes corresponding to ADR6, ADR11-2 and ADR12-2 was reduced in the presence of auxin, but the decrease was small relative to the decrease in the cytoplasmic levels of these mRNAs, in response to auxin. A comparative analysis of the influence of auxin on in vitro transcription and steady state RNA levels corresponding to these ADR cDNAs suggests that the decrease in rate of transcription due to auxin is not enough to account for the auxin-induced decrease in the steady state levels. Northern analysis showed developmental and organ/tissue-specific response of these ADR genes. Furthermore, the expression of the genes corresponding to ADR6 and ADR12-1 appears to be up-regulated by light, whereas the gene corresponding to ADR11 appears to be down-regulated by light.
Asunto(s)
Genes de Plantas/genética , Glycine max/genética , Ácidos Indolacéticos/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Regulación hacia Abajo/genética , Luz , Datos de Secuencia Molecular , Familia de Multigenes , Especificidad de Órganos , Poli A/metabolismo , ARN Mensajero/metabolismo , Transcripción Genética/fisiología , Regulación hacia Arriba/genéticaRESUMEN
The promoter region of a soybean auxin-responsive gene, GmAux28, was analyzed to identify protein-binding DNA sequences that may be involved in regulation of expression. Using DNase I footprinting and gel mobility shift assays, multiple regions of interaction, including eight major protein-binding sites, were observed in the GmAux28 gene. Two sequence motifs, TGACGACA and TCCACGTGTC, related to as-1/Hex and G-box elements, respectively, found in several plant promoters, were identified. Four distinct A/T-rich domains were identified; such A/T-rich domains appear to modulate, but not to specify, the expression of many genes. Two new sequence motifs, delta-1 (D1) and delta-4 (D4) were also identified. D1 and D4 share a very similar core sequence, TAGTxxCTGT and TAGTxCTGT, respectively. In gel mobility shift analyses, D1 and D4 elements exhibit a complex interaction of binding proteins. The GmAux22 promoter also contains D1-related elements which compete with the GmAux28 elements. Sequence comparisons have identified D1/D4-like sequences in several other auxin-responsive genes suggesting the possible importance of D1/D4 and the respective binding proteins in the regulation of expression of these genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Genes de Plantas , Glycine max/genética , Ácidos Indolacéticos/fisiología , Regiones Promotoras Genéticas , Secuencia de Bases , Sitios de Unión , ADN/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismoRESUMEN
Three related gene families of low-molecular-weight (LMW) heat shock proteins (HSPs) have been characterized in plants. We describe a fourth LMW HSP family, represented by PsHSP22.7 from Pisum sativum and GmHSP22.0 from Glycine max, and demonstrate that this family of proteins is endomembrane localized. PsHSP22.7 and GmHSP22.0 are 76.7% identical at the amino acid level. Both proteins have amino-terminal signal peptides and carboxyl-terminal sequences characteristic of endoplasmic reticulum (ER) retention signals. The two proteins closely resemble class I cytoplasmic LMW HSPs, suggesting that they evolved from the cytoplasmic proteins through the addition of the signal peptide and ER retention motif. The endomembrane localization of these proteins was confirmed by cell fractionation. The polypeptide product of PsHSP22.7 mRNA was processed to a smaller-M(r) form by canine pancreatic microsomes; in vivo, GmHSP22.0 polysomal mRNA was found to be predominantly membrane bound. In vitro-processed PsHSP22.7 corresponded in mass and pI to one of two proteins detected in ER fractions from heat-stressed plants by using anti-PsHSP22.7 antibodies. Like other LMW HSPs, PsHSP22.7 was observed in higher-molecular-weight structures with apparent masses of between 80 and 240 kDa. The results reported here indicate that members of this new class of LMW HSPs are most likely resident ER proteins and may be similar in function to related LMW HSPs in the cytoplasm. Along with the HSP90 and HSP70 classes of HSPs, this is the third category of HSPs localized to the ER.
Asunto(s)
Fabaceae/metabolismo , Proteínas de Choque Térmico/metabolismo , Plantas Medicinales , Secuencia de Aminoácidos , Compartimento Celular , Clonación Molecular , Retículo Endoplásmico/metabolismo , Fabaceae/genética , Fabaceae/ultraestructura , Genes de Plantas , Proteínas de Choque Térmico/genética , Calor , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Alineación de SecuenciaRESUMEN
The expression patterns of three members of a gene family that encodes proline-rich proteins in soybean (SbPRPs) were examined using in situ hybridization experiments. In most instances, the expression of SbPRP genes was intense in a limited number of cell types of a particular organ. SbPRP1 RNA was localized in several cell types of soybean hypocotyls, including cells within the phloem and xylem. SbPRP1 expression increased within epidermal cells in the elongating and mature regions of the hypocotyl; expression was detected also in lignified cells surrounding the hilum of mature seeds. SbPRP2 RNA was present in cortical cells and in the vascular tissue of the hypocotyl, especially cells of the phloem. This gene was expressed also in the inner integuments of the mature seed coat. SbPRP3 RNA was localized specifically to the endodermoid layer of cells surrounding the stele in the elongating region of the hypocotyl, as well as in the epidermal cells of leaves and cotyledons. These data show that members of this gene family exhibit cell-specific expression. The members of the SbPRP gene family are expressed in different types of cells and in some cell types that also express the glycine-rich protein or hydroxyproline-rich glycoprotein classes of genes.
Asunto(s)
Glycine max/genética , Biosíntesis de Péptidos , Péptidos , Proteínas de Plantas/biosíntesis , Histocitoquímica/métodos , Familia de Multigenes , Hibridación de Ácido Nucleico , Dominios Proteicos Ricos en Prolina , ARN Mensajero/aislamiento & purificación , Distribución TisularRESUMEN
A full-length cDNA encoding a heat shock protein (hsp) belonging to the 83 to 90 kilodalton hsp family of Arabidopsis thaliana has been isolated and sequenced. Truncated cDNA clones were isolated by nucleic acid hybridization to a truncated soybean HSP83 cDNA probe and a fragment generated from a Drosophila HSP83 gene. A single strand DNA vector/primer based extension procedure was employed to obtain the full-length cDNA. The level of transcripts homologous to this cDNA (AtHS83) is low in 2-week-old Arabidopsis plants but is rapidly enhanced by elevated temperatures. DNA sequence comparison between this cDNA and hsp83-90 sequences from human, yeast and Drosophila reveal amino acid identities of 63 to 69%, typical identities for interspecies comparisons between hsp83 to 90 kilodalton proteins. Genomic DNA blot analysis performed with probes derived from AtHS83 indicate the presence of a HSP83 gene family estimated to be comprised of at least three genes.
RESUMEN
The transcriptional response of soybean (Glycine max) seedlings during heat shock (HS) was investigated under two different treatment regimes. During prolonged heat treatment at 40 degrees C, active transcription of the HS genes (as measured by "runoff" transcription assays) occurs only during the first few hours. Nonetheless, mRNAs for these genes are present at relatively high abundance even after 9 hours of exposure to 40 degrees C. Because HS mRNAs have a fairly short half-life (less than 3 hours) at 28 degrees C, these results indicate that HS mRNAs are inherently more stable at 40 degrees C. During a second type of heat treatment regime-short pulses of high (45 degrees C) heat followed by 1 to 2 hours at 28 degrees C-transcription of HS genes is comparable to that achieved at 40 degrees C for the first few hours, even though the tissue is maintained at non-HS temperatures. The transcriptional responses to these two different heat treatments indicate that regulatory controls for the transcription of the HS genes must involve more than a simple sensing of ambient temperature, since transcription of these genes can be turned off at 40 degrees C (in the case of prolonged exposure) and can continue at 28 degrees C (following a short, severe heat treatment). Additional results demonstrate that the response of soybean seedlings to a particular HS depends on their prior exposure to heat; seedlings given a preheat treatment (that is known to induce thermotolerance) respond more moderately to a short heat pulse at 45 degrees C. Overall, this research indicates that plants have mechanisms for both monitoring the severity of changes in temperature and for measuring the magnitude and duration of the stress. Such information is then used to regulate the plant's response to heat both transcriptionally and posttranscriptionally.
RESUMEN
Further characterization of a proline-rich cell wall protein gene family from soybean (Glycine max (L.) Merr) has been accomplished by the isolation and sequence analysis of two additional genes, SbPRP2 and SbPRP3, which encode mRNAs of 1050 and 650 nucleotides in length, respectively. Like the proline-rich protein gene, SbPRP1, which was previously reported (Hong, J. C., Nagao, R. T., and Key, J. L. (1987) J. Biol. Chem. 262, 8367-8376), these two SbPRP genes encode proteins having a signal peptide sequence and repeats of Pro-Pro-Val-Tyr-Lys. The SbPRP2 gene encodes a protein of 26 kDa which contains a perfect alternating repeat of Pro-Pro-Val-Tyr-Lys and Pro-Pro-Val-Glu-Lys. The SbPRP3 encodes a 10-kDa protein which also contains Pro-Pro-Val-Tyr-Lys as a major amino acid repeat, but the overall amino acid sequence of this protein is more variable than that of SbPRP1 and SbPRP2. RNA blot analyses have demonstrated that there are marked differences in the pattern of expression of each SbPRP in various soybean tissues. In contrast, sequence analysis reveals that the SbPRP genes contain a high degree of sequence conservation. Nucleotide sequence homology extends 90 to 100 base pairs upstream of the transcription initiation site and includes typical CAT and TATA sequences. Approximately 80 base pairs of the 3'-noncoding sequence around the polyadenylation signal is also highly conserved. Therefore, the DNA sequence upstream of the 5'-conserved region is presumed to contain cis-elements accounting for the developmental and tissue specificity of gene expression. While the pentameric repeat structures occur in all SbPRP genes, the encoded proteins are predicted to be different in several features including basicity, substitutions of tyrosine and glutamic acid in the repeat, and the size of the mature protein.
Asunto(s)
Glycine max/genética , Familia de Multigenes , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Pared Celular/metabolismo , Clonación Molecular , Sondas de ADN , Biblioteca de Genes , Genes de Plantas , Datos de Secuencia Molecular , Conformación Proteica , ARN Mensajero/genética , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
Previously, we reported the characterization of a developmentally regulated proline-rich cell wall protein (SbPRP1) gene of soybean; the encoded protein is represented by a consensus amino acid repeat structure of Pro-Pro-Val-Tyr-Lys [Hong, J.C., Nagao, R.T., and Key, J.L. (1987). J. Biol. Chem. 262, 8367-8376]. Two other closely related members of this family of proline-rich protein (PRP) genes (SbPRP2 and SbPRP3), which differ from the extensin family of cell wall proteins, have been characterized (J.C. Hong, R.T. Nagao, and J.L. Key, unpublished results). Here we report studies on the regulation of expression of this gene family during soybean development by analyzing various plant organs, including leaves, stems, and roots of etiolated seedlings and light-grown plants, as well as young and mature pods, seed coats, and cotyledons. These organs were tested at different stages of development (young and fully mature tissue). Although a high level of sequence homology is observed at the nucleotide and amino acid sequence level among these three PRP genes/proteins, there are marked differences in the patterns of expression of each gene in different plant organs and organ regions. SbPRP1 is highly expressed and is the predominant form of PRP transcript in the mature hypocotyl, root, and immature seed coat. SbPRP2 is the major form of PRP in the apical hypocotyl and young suspension culture cells. SbPRP3 is the major form of PRP gene expressed in aerial parts; it is highly expressed in leaves, although no expression is detected in the roots.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Pared Celular/metabolismo , Regulación de la Expresión Génica , Glycine max/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Regulación de la Expresión Génica/efectos de la radiación , Luz , Datos de Secuencia Molecular , Familia de Multigenes , Especificidad de Órganos/genética , Semillas/genética , Glycine max/crecimiento & desarrollo , Glycine max/metabolismoRESUMEN
The auxin-regulated expression of two poly(A)+ mRNAs in soybean hypocotyl was demonstrated by cloning of the cDNAs and Northern blot hybridization analyses (Walker, J.C., and Key, J.L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7185-7189). The corresponding genes, designated Aux28 and Aux22, have been isolated, and the cDNAs and genes have been sequenced. The Aux28 and Aux22 genes are present at one to two copies per haploid genome, contain four and two introns, and encode hydrophilic proteins of 26.8 and 21.5 kDa, respectively. Although the cDNAs were isolated independently and do not cross-hybridize under stringent hybridization conditions, the protein coding sequences of the two cDNAs have several colinear regions of high homology at the nucleic acid (77-80%) and the amino acid (80-100%) levels; together these regions constitute approximately a third of the protein coding sequences of the cDNAs. These data, together with genomic Southern blot hybridization analysis and hybrid-select translations of mRNAs homologous to the cDNAs, show that these genes belong to two related multigene families. We have identified two sequences, TGATAAAAG and GGCAGCATGCA, that occur at similar distances upstream of the transcription start site in each gene, and the spacing between these two elements is essentially identical in the two genes. The possible significance of these sequences is under evaluation.
Asunto(s)
Genes , Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Enzimas de Restricción del ADN , Genes Homeobox , Datos de Secuencia Molecular , ARN Mensajero/genética , Glycine max/genética , Transcripción GenéticaRESUMEN
We determined the DNA sequence and mapped the corresponding transcripts of a genomic clone containing the Gmhsp26-A gene of soybean. This gene is homologous to the previously characterized cDNA clone pCE54 (E. Czarnecka, L. Edelman, F. Schöffl, and J. L. Key, Plant Mol. Biol. 3:45-58, 1984) and is expressed in response to a wide variety of physiological stresses including heat shock (HS). S1 nuclease mapping of transcripts and a comparison of the cDNA sequence with the genomic sequence indicated the presence of a soybean seedlings with either CdCl2 or CuSO4. Analysis of the 5' termini of transcripts indicated the presence of one major and at least two minor start sites. In each case, initiation occurred 27 to 30 base pairs downstream from a TATA-like motif, and thus each initiation site appears to be promoted by the activity of a separate subpromoter. The three subpromoters are all associated with sequences showing low homology to the HS consensus element of Drosophila melanogaster HS genes and are differentially induced in response to various stresses. Within the carboxyl-terminal half of the protein, hydropathy analysis of the deduced amino acid sequence indicated a high degree of relatedness to the small HS proteins. A comparison of the primary amino acid sequence of hsp26-A with sequences of the small HS proteins suggested that this stress protein is highly diverged and may therefore be specialized for stress adaptation in soybean.