ADAP is a possible negative regulator of glucosinolate biosynthesis in Arabidopsis thaliana based on clustering and gene expression analyses.

Glucosinolates (GSLs) are plant secondary metabolites consisting of sulfur and nitrogen, commonly found in Brassicaceae crops, such as Arabidopsis thaliana. These compounds are known for their roles in plant defense mechanisms against pests and pathogens. 'Guilt-by-association' (GBA) approach predicts genes encoding proteins with similar function tend to share gene expression pattern generated from high throughput sequencing data. Recent studies have successfully identified GSL genes using GBA approach, followed by targeted verification of gene expression and metabolite data. Therefore, a GSL co-expression network was constructed using known GSL genes obtained from our in-house database, SuCComBase. DPClusO was used to identify subnetworks of the GSL co-expression network followed by Fisher's exact test leading to the discovery of a potential gene that encodes the ARIA-interacting double AP2-domain protein (ADAP) transcription factor (TF). Further functional analysis was performed using an effective gene silencing system known as CRES-T. By applying CRES-T, ADAP TF gene was fused to a plant-specific EAR-motif repressor domain (SRDX), which suppresses the expression of ADAP target genes. In this study, ADAP was proposed as a negative regulator in aliphatic GSL biosynthesis due to the over-expression of downstream aliphatic GSL genes (UGT74C1 and IPMI1) in ADAP-SRDX line. The significant over-expression of ADAP gene in the ADAP-SRDX line also suggests the behavior of the TF that negatively affects the expression of UGT74C1 and IPMI1 via a feedback mechanism in A. thaliana.

Repression domains of class II ERF transcriptional repressors share an essential motif for active repression.

We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif (L)/(F)DLN(L)/(F)(x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.

Regulation of ethylene-induced transcription of defense genes.

Ethylene-induced gene expression has been studied in systems in which the biosynthesis of ethylene is stimulated during developmental process such as ripening of fruit, senescence of flower petals, or during pathogen infection. Functional analysis of the promoters of these genes revealed that the ethylene-responsive cis-elements of fruit ripening genes and senescence genes differed from that of defense genes whose expression is induced by ethylene in response to pathogen infection. The ethylene-responsive element identified as the GCC box (AGCCGCC) is commonly found in the promoter region of the ethylene-inducible defense genes. The ethylene responsive element binding factors that interact with the GCC box were demonstrated to be the transcription factors, which respond to extracellular signals to modulate GCC box-mediated gene expression positively or negatively.

Characterization of gene expression of NsERFs, transcription factors of basic PR genes from Nicotiana sylvestris.

Three genes of NsERFs (EREBPs), transcription factors for GCC box of basic PR genes, were isolated from Nicotiana sylvestris. Analyses of transgenic tobacco carrying the NsERF promoter::GUS genes showed that expression of all NsERF genes in leaves was induced by ethylene. Sequence analyses indicated that the 5'-upstream region of NsERF3 gene has the GCC box. In contrast, the promoter regions of NsERF2 and 4 have no GCC box, whereas NsERF2 has a putative EIN3 binding site. Tissue/cell specific expression is also discussed.

Three ethylene-responsive transcription factors in tobacco with distinct transactivation functions.

Ethylene-responsive factors (ERFs) have conserved DNA-binding domains and interact directly with the GCC box in the ethylene-responsive element that is necessary and sufficient for the regulation of transcription by ethylene. ERFs were shown to be localized to nucleus in transient transfection experiments. Transient expression assays using tobacco protoplasts and a heterologous system in yeast were used to examine the transactivation functions of ERFs. ERF2 and ERF4 enhanced the GCC box-mediated transcription of a reporter gene in tobacco protoplasts. When fused to the DNA-binding domain of yeast GAL4, a carboxy-terminal region of ERF2, as well as both amino-terminal and carboxy-terminal regions of ERF4, functioned as a transactivation domain in tobacco protoplasts. The amino-terminal regions of ERF2 and ERF4 functioned as transactivation domains in yeast. In contrast to ERF2 and ERF4, ERF3 reduced the transcription of the reporter gene in tobacco protoplasts, indicating that ERF3 functions as a repressor. Thus, it appears that ERFs exert their regulatory functions in different ways, with ERF2 and ERF4 being activators and ERF3 being a repressor of transcription.

Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression.

Ethylene-responsive element binding factors (ERFs) are members of a novel family of transcription factors that are specific to plants. A highly conserved DNA binding domain known as the ERF domain is the unique feature of this protein family. To characterize in detail this family of transcription factors, we isolated Arabidopsis cDNAs encoding five different ERF proteins (AtERF1 to AtERF5) and analyzed their structure, DNA binding preference, transactivation ability, and mRNA expression profiles. The isolated AtERFs were placed into three classes based on amino acid identity within the ERF domain, although all five displayed GCC box-specific binding activity. AtERF1, AtERF2, and AtERF5 functioned as activators of GCC box-dependent transcription in Arabidopsis leaves. By contrast, AtERF3 and AtERF4 acted as repressors that downregulated not only basal transcription levels of a reporter gene but also the transactivation activity of other transcription factors. The AtERF genes were differentially regulated by ethylene and by abiotic stress conditions, such as wounding, cold, high salinity, or drought, via ETHYLENE-INSENSITIVE2 (EIN2)-dependent or -independent pathways. Cycloheximide, a protein synthesis inhibitor, also induced marked accumulation of AtERF mRNAs. Thus, we conclude that AtERFs are factors that respond to extracellular signals to modulate GCC box-mediated gene expression positively or negatively.

A tobacco gene encoding a novel basic class II chitinase: a putative ancestor of basic class I and acidic class II chitinase genes.

Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal signal peptide, followed by a 245-amino acid chitinolytic domain. Although the predicted mature protein is basic and shows greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases. Therefore, this gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1. Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon. Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations. Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms. Possible mechanisms for chitinase gene evolution are discussed.

Unique mode of GCC box recognition by the DNA-binding domain of ethylene-responsive element-binding factor (ERF domain) in plant.

Ethylene-responsive element-binding proteins (EREBPs)have novel DNA-binding domains (ERF domains), which are widely conserved in plants, and interact specifically with sequences containing AGCCGCC motifs (GCC box). Deletion experiments show that some flanking region at the N terminus of the conserved 59-amino acid ERF domain is required for stable binding to the GCC box. Three ERF domain-containing fragments of EREBP2, EREBP4, and AtERF1 from tobacco and Arabidopsis, bind to the sequence containing the GCC box with a high binding affinity in the pM range. The high affinity binding is conferred by a monomeric ERF domain fragment, and DNA truncation experiments show that only 11-base pair DNA containing the GCC box is sufficient for stable ERF domain interaction. Systematic DNA mutation analyses demonstrate that the specific amino acid contacts are confined within the 6-base pair GCCGCC region of the GCC box, and the first G, the fourth G, and the sixth C exhibit highest binding specificity common in all three ERF domain-containing fragments studied. Other bases within the GCC box exhibit modulated binding specificity varying from protein to protein, implying that these positions are important for differential binding by different EREBPs. The conserved N-terminal half is likely responsible for formation of a stable complex with the GCC box and the divergent C-terminal half for modulating the specificity.

A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA.

The 3D solution structure of the GCC-box binding domain of a protein from Arabidopsis thaliana in complex with its target DNA fragment has been determined by heteronuclear multidimensional NMR in combination with simulated annealing and restrained molecular dynamic calculation. The domain consists of a three-stranded anti-parallel beta-sheet and an alpha-helix packed approximately parallel to the beta-sheet. Arginine and tryptophan residues in the beta-sheet are identified to contact eight of the nine consecutive base pairs in the major groove, and at the same time bind to the sugar phosphate backbones. The target DNA bends slightly at the central CG step, thereby allowing the DNA to follow the curvature of the beta-sheet.

Identification of an ethylene-responsive region in the promoter of a tobacco class I chitinase gene.

The Chn48 gene is a representative of a family of tobacco class I basic chitinase genes, and the expression is induced by the stress hormone ethylene. To investigate the molecular basis for transcriptional regulation by ethylene we have examined the Chn48 promoter to identify cis-elements and trans-acting factors that are involved in the chitinase gene expression. In transgenic tobacco plants, a chimeric gene construct containing a 2 kb Chn48 promoter fused to a beta-glucuronidase reporter gene was induced by ethylene in leaf tissues. Deletion analysis indicated that a positive ethylene-responsive region is located between nucleotides -503 and -358 relative to the transcription initiation site. This 146 bp sequence was found to confer ethylene-responsive reporter gene expression when inserted in either orientation upstream of the heterologous promoter, indicating that the sequence functions as a regulatory enhancer. The ethylene-responsive region contains two copies of a GCC-box (TAAGAGCCGCC), which is conserved in a number of ethylene-responsive defense genes. The sequences within this ethylene-responsive region that are necessary for ethylene-responsive transcription were further localized to the 71 bp sequence between positions -480 and -410 containing two copies of the GCC-box by loss-of-function analysis. Gel mobility-shift experiments showed the presence of leaf nuclear factors that interact with the DNA sequences included in the ethylene-responsive region.

Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element.

We demonstrated that the GCC box, which is an 11-bp sequence (TAAGAGCCGCC) conserved in the 5' upstream region of ethylene-inducible pathogenesis-related protein genes in Nicotiana spp and in some other plants, is the sequence that is essential for ethylene responsiveness when incorporated into a heterologous promoter. Competitive gel retardation assays showed DNA binding activities to be specific to the GCC box sequence in tobacco nuclear extracts. Four different cDNAs encoding DNA binding proteins specific for the GCC box sequence were isolated, and their products were designated ethylene-responsive element binding proteins (EREBPs). The deduced amino acid sequences of EREBPs exhibited no homology with those of known DNA binding proteins or transcription factors; neither did the deduced proteins contain a basic leucine zipper or zinc finger motif. The DNA binding domain was identified within a region of 59 amino acid residues that was common to all four deduced EREBPs. Regions highly homologous to the DNA binding domain of EREBPs were found in proteins deduced from the cDNAs of various plants, suggesting that this domain is evolutionarily conserved in plants. RNA gel blot analysis revealed that accumulation of mRNAs for EREBPs was induced by ethylene, but individual EREBPs exhibited different patterns of expression.

The effect of sequences with high AU content on mRNA stability in tobacco.

Little is known about the mechanisms that target transcripts for rapid degradation in plants. In mammalian cells, sequences with a high AU content and multiple AUUUA motifs have been shown to cause mRNA instability when present in the 3' untranslated regions of several transcripts. This precedent, coupled with the poor accumulation of AU-rich foreign transcripts in plants (e.g., BT-toxin mRNAs), prompted us to test whether AU sequences could destabilize transcripts in tobacco. To address this question, we made a set of constructs containing sequences with high AU content inserted into the 3' untranslated regions of reporter genes. The stability of the corresponding transcripts was then assayed in stably transformed cell lines of tobacco. These experiments showed that a 60-base sequence containing 11 copies of the AUUUA motif (AUUUA repeat) markedly destabilized a beta-glucuronidase reporter transcript compared to a no-insert control or a 60-base spacer sequence (GC control). Another sequence with an identical A+U content had little effect. The same results were obtained when each sequence was assayed within the 3' untranslated region of a beta-globin reporter transcript. In regenerated transgenic plants, the AUUUA repeat decreased the accumulation of the beta-globin transcript by approximately 14-fold, compared to the GC control. Taken together, our results indicate that the AUUUA repeat is recognized as an instability determinant in plant cells and that the effect is due to the sequence of the element, not simply to the high AU content.

DST sequences, highly conserved among plant SAUR genes, target reporter transcripts for rapid decay in tobacco.

DST elements are highly conserved sequences located in the 3' untranslated regions (UTRs) of a set of unstable soybean transcripts known as the small auxin-up RNAs (SAURs). To test whether DST sequences could function as mRNA instability determinants in plants, a model system was developed to facilitate the direct measurement of mRNA decay rates in stably transformed cells of tobacco. Initial experiments established that the chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) transcripts degraded with similar half-lives in this system. In addition, their decay kinetics mirrored the apparent decay kinetics of the corresponding transcripts produced in transgenic plants under the control of a regulated promoter (Cab-1). The model system was then used to measure the decay rates of GUS reporter transcripts containing copies of the DST sequence inserted into the 3'UTR. An unmodified CAT gene introduced on the same vector served as the internal reference. These experiments and a parallel set utilizing a beta-globin reporter gene demonstrated that a synthetic dimer of the DST sequence was sufficient to destabilize both reporter transcripts in stably transformed tobacco cells. The decrease in transcript stability caused by the DST sequences in cultured cells was paralleled by a coordinate decrease in transcript abundance in transgenic tobacco plants. The implications of these results for the potential function of DST sequences within the SAUR transcripts are discussed.

Structure and expression of a tobacco beta-1,3-glucanase gene.

We determined the primary structure of a tobacco beta-1,3-glucanase gene. The beta-1,3-glucanase gene has a single large intron, and the intron separates coding regions of the signal peptide and the mature enzyme. Analysis of the 5'-flanking region sequence revealed an 11 bp GC-rich element with perfect homology to the putative regulatory sequence of tobacco chitinase genes. RNA blot analysis showed that levels of mRNAs of beta-1,3-glucanase and chitinase are coordinately increased in response to ethylene and salicylic acid. Accumulation of beta-1,3-glucanase mRNA in suspension-cultured cells is rapidly induced at late logarithmic growth phase. Members of the tobacco beta-1,3-glucanase gene families are classified into two subfamilies. One of the subfamilies appeared to be transcriptionally inactive.

In vivo RNA transcript-releasing plasmid possessing a universal pseudo-terminator by means of artificial ribozymes.

RNA transcript-releasing plasmid has been constructed by means of artificial hammerhead ribozymes. In this specific construct of pGENE8459v3 the ribozyme targeted for SFL1 gene (a yeast suppressor gene for flocculation) was fused between two other ribozymes called 5'-processing and 3'-processing ribozymes. Since the "Ribozyme for SFL1" portion (cassette) can be replaced by other RNA sequences, it is now possible to produce any RNAs with defined 5'- and 3'- ends.