The transcript abundance of GmGT-2, a new member of the GT-2 family of transcription factors from soybean, is down-regulated by light in a phytochrome-dependent manner.

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.

STF1 is a novel TGACG-binding factor with a zinc-finger motif and a bZIP domain which heterodimerizes with GBF proteins.

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.

Tissue-Type-Specific Heat-Shock Response and Immunolocalization of Class I Low-Molecular-Weight Heat-Shock Proteins in Soybean.

A monospecific polyclonal antibody was used to study the tissue-type specificity and intracellular localization of class I low-molecular-weight (LMW) heat-shock proteins (HSPs) in soybean (Glycine max) under different heat-shock regimes. In etiolated soybean seedlings, the root meristematic regions contained the highest levels of LMW HSP. No tissue-type-specific expression of class I LMW HSP was detected using the tissue-printing method. In immunolocalization studies of seedlings treated with HS (40[deg]C for 2 h) the class I LMW HSPs were found in the aggregated granular structures, which were distributed randomly in the cytoplasm and in the nucleus. When the heat shock was released, the granular structures disappeared and the class I LMW HSPs became distributed homogeneously in the cytoplasm. When the seedlings were then given a more severe heat shock following the initial 40[deg]C -> 28[deg]C treatment, a large proportion of the class I LMW HSPs that originally localized in the cytoplasm were translocated into the nucleus and nucleolus. Class I LMW HSPs may assist in the resolubilization of proteins denatured or aggregated by heat and may also participate in the restoration of organellar function after heat shock.

Induction and Regulation of Heat-Shock Gene Expression by an Amino Acid Analog in Soybean Seedlings.

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.

Molecular characterization of cDNAs encoding low-molecular-weight heat shock proteins of soybean.

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.

Isolation of two soybean G-box binding factors which interact with a G-box sequence of an auxin-responsive gene.

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.

A soybean 101-kD heat shock protein complements a yeast HSP104 deletion mutant in acquiring thermotolerance.

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.

Isolation and characterization of three soybean extensin cDNAs.

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.

Expression of the Arabidopsis AtAux2-11 auxin-responsive gene in transgenic plants.

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.

Regulatable endogenous production of cytokinins up to 'toxic' levels in transgenic plants and plant tissues.

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.

Identification of protein-binding DNA sequences in an auxin-regulated gene of soybean.

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.

Isolation and characterization of three families of auxin down-regulated cDNA clones.

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.

Localization of small heat shock proteins to the higher plant endomembrane system.

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.

Patterns of soybean proline-rich protein gene expression.

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.

Isolation and characterization of a soybean hsp70 gene.

Soybean, like many other organisms, responds to an increase in growth temperature by producing a set of new proteins, heat shock proteins. The heat shock proteins have been classified into several categories according to their molecular weight. Data are presented on the isolation, sequence characterization, and expression of a 70 kDa heat shock protein gene from soybean. A cDNA clone was isolated using a Drosophila hsp70 clone as a heterologous probe, and the cDNA was used for isolation of the soybean gene corresponding to the cDNA. The structure of this soybean is very similar to the hsp70 genes from other organisms. It has several sequences in the 5' untranscribed region that are similar to the well characterized heat shock consensus element found in other organisms. These heat shock consensus elements have the expected position relative to the start of transcription. Unlike hsp70-like genes previously isolated from other plants, this gene does not have an intron. This protein shows high amino acid sequence similarity to other hsp70 proteins from such diverse organisms as Drosophila, rat, and Xenopus. This soybean gene is only expressed during heat shock. In addition to the hsp70 gene isolated here, there is evidence for many other hsp70-like genes in soybean.

Sequence and Expression of a HSP83 from Arabidopsis thaliana.

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.

Regulation of the heat shock response in soybean seedlings.

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.

Structure and expression of two auxin-inducible genes from Arabidopsis.

Two genes from Arabidopsis thaliana related to the auxin-inducible Aux28 and Aux22 genes of soybean have been isolated. These genes belong to a small multi-gene family and are similar to the soybean Aux gene family in the sequence of the predicted proteins, intron/exon locations, and auxin-enhanced expression of their transcripts. Application of auxin to 8-day old Arabidopsis plants, 4-day old etiolated seedlings, and suspension culture cells all resulted in enhanced Aux transcript levels. Comparison of the promoter sequences from the soybean and Arabidopsis genes yielded no significant sequence conservation; however, three regions of near sequence identity are present between the two Arabidopsis Aux genes.