G-box Element Research Articles

In vivo binding of hot pepper bZIP transcription factor CabZIP1 to the G-box region of pathogenesis-related protein 1 promoter

We find that salicylic acid and ethephon treatment in hot pepper increases the expression of a putative basic/leucine zipper (bZIP) transcription factor gene, CabZIP1. CabZIP1 mRNA is expressed ubiquitously in various organs. The green fluorescent protein-fused transcription factor, CabZIP1::GFP, can be specifically localized to the nucleus, an action that is consistent with the presence of a nuclear localization signal in its protein sequence. Transient overexpression of the CabZIP1 transcription factor results in an increase in PR-1 transcripts level in Nicotiana benthamiana leaves. Using chromatin immunoprecipitation, we demonstrate that CabZIP1 binds to the G-box elements in native promoter of the hot pepper pathogenesis-related protein 1 ( CaPR-1) gene in vivo. Taken together, our results suggest that CabZIP1 plays a role as a transcriptional regulator of the CaPR-1 gene.

Functional Characterization of Phytochrome Interacting Factor 3 for the Arabidopsis thaliana Circadian Clockwork

Light, in a quality- and quantity-dependent fashion, induces nuclear import of the plant photoreceptors phytochromes and promotes interaction of these receptors with transcription factors including PHYTOCHROME INTERACTING FACTOR 3 (PIF3). PIF3 was shown to form in vitro a ternary complex with the G-box element of the promoters of LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and the Pfr conformer of phytochromes. CCA1 and LHY together with TIMING OF CAB EXPRESSION 1 (TOC1) constitute a transcriptional feed-back loop that is essential for a functional circadian clock in Arabidopsis. These findings led to the hypothesis that the PIF3-containing ternary complex regulates transcription of light-responsive genes and is involved in phototransduction to the central circadian clockwork. Here we report that (i) overexpression or lack of biologically functional PIF3 does not affect period length of rhythmic gene expression or red-light-induced resetting of the circadian clock and (ii) the transcription of PIF3 displays a low-amplitude circadian rhythm. We demonstrated previously that irradiation of etiolated seedlings induces rapid, phytochrome-controlled degradation of PIF3. Here we show that nuclear-localized PIF3 accumulates to relatively high levels by the end of the light phase in seedlings grown under diurnal conditions. Taken together, we show that (i) PIF3 does not play a significant role in controlling light input to and function of the circadian clockwork and (ii) a yet unknown mechanism limits phytochrome-induced degradation of PIF3 at the end of the day under diurnal conditions.

Methyl jasmonate induced expression of the tobacco putrescine N-methyltransferase genes requires both G-box and GCC-motif elements

Putrescine N-methyltransferase (PMT) catalyzes the first committed step of nicotine biosynthesis, converting putrescine into N-methylputrescine. A variety of chemical, environmental, and developmental cues have been implicated in its regulation. Here we have examined the differential expression of beta-glucuronidase (GUS) transgenes under the control of the transcriptional regulatory sequences of four distinct members of the NtPMT gene family from tobacco (Nicotiana tabacum L.). BY-2 cell cultures expressing various NtPMT promoter-GUS constructs were examined for their response to treatment with various combinations of methyl jasmonate (MeJA), auxin (AUX), and ethylene (ETH). All four NtPMT gene promoters examined were inducible by MeJA, although the extent of the induction varied dramatically, with the NtPMT1a promoter being the most responsive. High AUX levels in the cell growth media repressed NtPMT::GUS transgene expression and inhibited their MeJA-induced transcription. Treatment of BY-2 cells with ETH alone did not result in a significant alteration in NtPMT::GUS expression. However, similar to AUX, ETH treatment led to the suppression of MeJA-induced transcription. Detailed deletion analysis of the NtPMT1a gene promoter showed that as little as 111 bp upstream of the transcriptional start site were sufficient to confer MeJA-responsiveness. Deletion of a conserved G-box element (GCACGTTG) at -103 to -96 bp completely abolished MeJA-responsiveness. Further mutagenesis studies revealed that in addition to a functional G-box, MeJA-responsiveness of the NtPMT1a promoter also required a TA-rich region and a GCC-motif (TGCGCCC) located at -80 to -69 bp and -62 to -56 bp relative to the start site, respectively. A synthetic G-box tetramer (4 X syn G-box) fused to a -83 bp fragment from the NtPMT1a promoter (containing the TA-rich region, GCC-box, and TATA-box) displayed a 30-fold induction by MeJA treatment, whereas when the 4 X syn G-box was fused to a minimal (-46 bp) promoter fragment derived from the CaMV 35S gene, no induction by MeJA treatment was detected. Our results indicate that multiple intersecting signal transduction pathways and different transcriptional regulatory factors are involved in mediating JA-responsiveness of NtPMT expression in tobacco.

Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis.

Jasmonates (JA) are important regulators of plant defense responses that activate expression of many wound-induced genes including the tomato proteinase inhibitor II (pin2) and leucine aminopeptidase (LAP) genes. Elements required for JA induction of the LAP gene are all present in the -317 to -78 proximal promoter region. Using yeast one-hybrid screening, we have identified the bHLH-leu zipper JAMYC2 and JAMYC10 proteins, specifically recognizing a T/G-box AACGTG motif in this promoter fragment. Mutation of the G-box element decreases JA-responsive LAP promoter expression. Expression of JAMYC2 and JAMYC10 is induced by JA, with a kinetics that precedes that of the LAP or pin2 transcripts. JAMYC overexpression enhanced JA-induced expression of these defense genes in potato, but did not result in constitutive transcript accumulation. Using footprinting assays, an additional protected element was identified, located directly adjacent to the T/G-box motif. Mutation of this element abolishes JA response, showing that recognition of this duplicated element is also required for gene expression. Knockout mutants in the AtMYC2 homolog gene of Arabidopsis are insensitive to JA and exhibit a decreased activation of the JA-responsive genes AtVSP and JR1. Activation of the PDF1.2 and b-CHI, ethylene/JA-responsive genes, is, however, increased in these mutants. These results show that the JAMYC/AtMYC2 transcription factors function as members of a MYC-based regulatory system conserved in dicotyledonous plants with a key role in JA-induced defense gene activation.

Differential induction of ornithine decarboxylase (ODC) gene family members in transgenic tobacco (Nicotiana tabacum L. cv. Bright Yellow 2) cell suspensions by methyl-jasmonate treatment

Two nuclear genes (NtODC-1 and NtODC-2) encoding ornithine decarboxylase (ODC, EC 4.1.1.17) were characterized from tobacco (Nicotiana tabacum L., and their differential induction by methyl-jasmonate (MeJA) was analyzed. ODC transcript levels increased in BY-2 cells following treatment with MeJA, with the highest levels of transcript observed approximately 5 h post-treatment. Analysis of transgenic BY-2 cell lines expressing a β-glucuronidase (GUS) reporter gene under the control of either the NtODC-1 or NtODC-2 gene promoter revealed that expression of the NtODC-1::GUS transgene was highly induced (8-fold) by treatment with MeJA, whereas the NtODC-2::GUS transgene showed little MeJA induced GUS expression. Removal of auxin (AUX, 2,4-D) from the cell growth media led to an increase in NtODC-1 and NtODC-2 transcript levels, whereas high levels of 2,4-D repressed overall ODC gene expression and suppressed the MeJA-induced expression of NtODC-1. Deletion analysis identified an upstream region (−924 to −1424 bp relative to the start site of transcription) of the NtODC-1 promoter that confers MeJA-responsiveness. No similar region was identified in the NtODC-2 promoter. Sequence analysis revealed that the NtODC-1 promoter fragment contains both G-box and TGACG-motif elements similar to those found in other MeJA-responsive genes. Our findings are discussed in terms of potential factors mediating different phytohormone-activated signal transduction pathways in the formation of nicotine and related compounds in tobacco.

A sugar beet chlorophyll a/b binding protein promoter void of G-box like elements confers strong and leaf specific reporter gene expression in transgenic sugar beet

BackgroundModification of leaf traits in sugar beet requires a strong leaf specific promoter. With such a promoter, expression in taproots can be avoided which may otherwise take away available energy resources for sugar accumulation.ResultsSuppression Subtractive Hybridization (SSH) was utilized to generate an enriched and equalized cDNA library for leaf expressed genes from sugar beet. Fourteen cDNA fragments corresponding to thirteen different genes were isolated. Northern blot analysis indicates the desired tissue specificity of these genes. The promoters for two chlorophyll a/b binding protein genes (Bvcab11 and Bvcab12) were isolated, linked to reporter genes, and transformed into sugar beet using promoter reporter gene fusions. Transient and transgenic analysis indicate that both promoters direct leaf specific gene expression. A bioinformatic analysis revealed that the Bvcab11 promoter is void of G-box like regulatory elements with a palindromic ACGT core sequence. The data indicate that the presence of a G-box element is not a prerequisite for leaf specific and light induced gene expression in sugar beet.ConclusionsThis work shows that SSH can be successfully employed for the identification and subsequent isolation of tissue specific sugar beet promoters. These promoters are shown to drive strong leaf specific gene expression in transgenic sugar beet. The application of these promoters for expressing resistance improving genes against foliar diseases is discussed.

CrMYC1, a Catharanthus roseus elicitor- and jasmonate-responsive bHLH transcription factor that binds the G-box element of the strictosidine synthase gene promoter.

A cDNA encoding a bHLH transcription factor was isolated by the yeast one-hybrid system from a Catharanthus roseus cDNA library using the G-box element of the Strictosidine synthase gene promoter as bait. The corresponding protein (named CrMYC1) was shown to bind specifically to the G-box in yeast. In C. roseus suspension cells CrMYC1 mRNA levels are induced by fungal elicitor and jasmonate suggesting that CrMYC1 may be involved in the regulation of gene expression in response to these signals.

Molecular analysis of flax 2S storage protein conlinin and seed specific activity of its promoter

The biochemical properties of conlinin, a 2S storage protein of flax ( Linum usitatissimum L.), are known but the gene encoding the protein has not been defined. Here, we report the identification of cDNA and genomic clones of two conlinin genes, conlinin1 ( cnl1) and conlinin2 ( cnl2). The cDNAs contain a single exon encoding a polypeptide containing a putative signal peptide and some features (conserved cysteine residues, glutamine-rich stretches, amino acid composition) characteristic of 2S storage proteins. The promoter regions of the two conlinin genes contain G-box and RY-like cis-acting elements. Northern analysis indicated that the genes are preferentially expressed in developing seeds with the maximal level at 20–35 d after flowering. Analysis of transgenic flax and Arabidopsis thaliana containing the GUS reporter gene under the control of the cnl1 promoter confirmed the seed specific pattern of expression.

Macroptilium yellow mosaic virus, a New Begomovirus Infecting Macroptilium lathyroides in Cuba

Macroptilium lathyroides (L) is a weed that is widely distributed in Cuba. Frequently, leaves show bright yellow mosaic symptoms, which suggest the incidence of a viral disease. Since begomovirus occurrence in Macroptilium lathyroides has been previously reported in other islands of the Caribbean (1,3), symptomatic plants from three distant places in Cuba (Havana, Villa Clara, and Camaguey), were collected and tested for the presence of begomoviruses. Plant DNA extracts were analyzed by Southern blot hybridization and polymerase chain reaction with two sets of degenerate primers (2). The presence of a bipartite begomovirus was evident through strong hybridization signals obtained with the DNA-A and DNA-B of Taino tomato mottle virus as probes at low stringency. Furthermore, 1.4-kb and 1.2-kb PCR amplified fragments were obtained with DNA-A degenerate primers, PAL1v1978-PAR1c715 and PAL1c1960-PAR1v722, respectively. Both PCR fragments from the samples from the three locations were cloned, and restriction fragment length polymorphism analysis of the 1.4-kb fragments were performed using PstI, EcoRI, HincII, XbaI and BglII. Restriction fragment patterns were the same for the three clones. The DNA-A sequence (GenBank Accession No. AJ344452) of the isolate from Villa Clara was compared with sequences available for other geminiviruses using CLUSTAL program. For the coat protein (CP) gene, the comparisons had the highest percentage of identity with various strains of Bean golden yellow mosaic virus (BGYMV, GenBank Accession Nos. AF173555, M91604, and L01635) (85 to 87% and 93 to 94%, nucleotide and amino acid sequences, respectively). For Rep gene (1,044 nt), the best percentages of identities were with BGYMV (81 to 82% and 80 to 82% nucleotide and amino acid sequences, respectively), Tomato leaf crumple virus (GenBank Accession No. AF101476) (78 and 81%, nucleotide and amino acid sequences, respectively), and Sida golden mosaic virus from Florida (GenBank Accession No. AF049336) (78 and 79%, nucleotide and amino acid sequences, respectively). Finally, the comparative analysis of the intergenic region (i.e. the common region plus the CP gene promoter) had the highest identity with BGYMV (56 to 55%) and Tomato severe rugose virus (GenBank Accession No. AY029750) (49%). Interestingly, this virus has in this region the three G-box elements that are characteristic of BGYMV but it differs in the Rep protein-binding iterative motif that is GGTGA instead of GGAGA, for BGYMV. These data indicate that this virus is a new begomovirus and the name of Macroptilium yellow mosaic virus (MaYMV) is proposed.

Commentary

A 45-bp proximal region of the rice glutelin promoter (−104/−60) containing two putative cis-elements, the AACA motif and GCN4 motifs, was fused to a truncated CaMV 35S promoter (−90/+9; −90Δ35S)/GUS. The 45-bp fragment specifically enhanced the promoter activity in endosperm tissue of transformed tobacco. A substitution mutation of the GCN4 motif reduced the promoter activity, whereas mutation of the AACA motif increased the activity in the embryo as well as in the endosperm. These results suggest that the GCN4 motif generally enhances the promoter activity but that the combination of the two motifs confers the endosperm specificity. Furthermore, the function of the two motifs was dependent on the orientation and/or distance from a G-box element in −90Δ35S, suggesting that synergistic interaction between the factors that recognize those motifs and the G-box element is important for transcriptional regulation.

The tobacco bZIP transcription factor BZI-1 binds to G-box elements in the promoters of phenylpropanoid pathway genes in vitro, but it is not involved in their regulation in vivo

Screening of a tobacco (Nicotiana tabacum) cDNA library resulted in the isolation of a clone encoding the bZIP transcription factor BZI-1. With respect to amino acid sequence, conservation of protein domains, genomic exon-intron structure and expression pattern, BZI-1 is closely related to CPRF2, OHP1/2, BLZ1 and REB, a group of bZIP proteins which have been described in a number of dicot and monocot species. BZI-1 exhibits the characteristics of a transcription factor. It binds to G-box and C-box cis-elements in vitro, it is localised in the nucleus, and the N-terminal region of BZI-1 functions as an activation domain in both yeast and plant cells. Since BZI-1-related transcription factors have been isolated from dicots by in vitro binding to G-box elements in the chalcone synthase ( CHS) promoter, it has been suggested that phenylpropanoid pathway genes, such as CHS and PAL (phenylalanine ammonia-lyase), are targets of these proteins in vivo. However, after infection with Pseudomonas syringae or Tobacco Mosaic Virus, no changes in pathogen-induced PAL expression were observed in transgenic plants expressing increased levels of BZI-1 or a dominant negative form of the protein, BZI-1-DeltaN. In contrast to the tissue-specific expression of CHS and PAL, BZI-1 was found to be ubiquitously expressed in tobacco plants. Furthermore, no changes in the tissue-specific expression of PAL or CHS were observed in plants that were transgenic for BZI-1-DeltaN. Expression of a VP16-BZI-1 fusion protein would be expected to result in constitutive activation of the BZI-1 target genes. However, tetracycline-dependent expression of a VP16-BZI-1 protein in tobacco plants did not result in activation of CHS or PAL. On the basis of these data, we conclude that the phenylpropanoid pathway genes analysed are not targets of BZI-1 in vivo. Thus, the pattern of in vitro DNA binding of transcription factors need not always reflect their in vivo function.

Reduction of G-box binding factor DNA binding activity, but not G-box binding factor abundance, causes the downregulation of RBCS2 expression during early tomato fruit development

The downregulation of RBCS2 promoter activity during tomato fruit development has been investigated by transient gene expression. A major drop in promoter activity occurs between 5 and 25 mm fruit diameter, corresponding to the late cell division to early cell enlargement phase. This drop is abolished by a mutation of the single G-box element necessary for high RBCS2 promoter activity in young tomato fruit. The G-box binding activity of fruit nuclear and total protein extracts drops concomitantly with the reduction of RBCS2 promoter activity while G-box binding factor expression is not affected. The data indicate that the developmental signal that downregulates the RBCS2 promoter acts on the regulation of DNA binding activity of constitutively expressed G-box binding factors.

A G-box element from the Catharanthus roseus strictosidine synthase (Str) gene promoter confers seed-specific expression in transgenic tobacco plants.

The enzyme encoded by the strictosidine synthase (Str) gene from Catharanthus roseus catalyses a key step in the biosynthesis of the pharmaceutically important terpenoid indole alkaloids. Str cDNA and genomic clones have already been isolated, allowing us to study the regulation of Str gene expression. Here we focus on the role of a putative cis-acting element, CACGTG, in the Str promoter. This sequence is known as a G-box, and functions as a transcription-regulating sequence in a number of other promoters. By means of electrophoretic mobility shift assays it was demonstrated that the Str G-box is capable of interacting with nuclear factors in tobacco and with the cloned tobacco G-box-binding factor TAF-1. Disruption of the Str G-box sequence by two single-nucleotide mutations prevented binding of factors, thereby demonstrating the specificity of the observed interactions. Functional analysis in transgenic tobacco plants demonstrated that these mutations also reduced the transcriptional activity of constructs containing tetramers of the Str G-box sequence. Expression directed by a tetramer of the Str G-box fused to a truncated promoter containing only a TATA box was confined to seeds and was found to increase during seed maturation. Thus, the Str G-box tetramer is able to direct seed-specific expression independently of other regulatory sequences. G-box-directed expression in leaves required the presence of an enhancer region from the cauliflower mosaic virus (CaMV) 35S promoter. The results indicate that the G-box needs to interact with other elements to drive expression in leaf, and that it can by itself confer seed-specific expression as a multimer. The fact that only some of the G-boxes found in different promoters serve as seed-specific elements indicates that sequences flanking the G-box determine the transcriptional activity in different tissues. Based on sequence comparisons we propose that the nucleotides at positions -4, -3, -2 and/or +4 are important in determining seed-specific expression.

A -308 deletion of the tomato LAP promoters is able to direct flower-specific and MeJA-induced expression in transgenic plants.

Tomato and potato leucine aminopeptidase (LAP) mRNAs are induced in response to mechanical wounding and the wound signal molecules, ABA and jasmonic acid. Here, we report the isolation of two LAP genes, LAP17.1A and LAP17.2, from tomato. Functional analysis in transgenic tomato and potato plants show that fusions of the corresponding 5' non-coding regions to the gusA gene are constitutively expressed in flowers and induced in leaves upon wounding or by treatment with methyl jasmonate (MeJA). Comparison of the 5' non-coding regions of the two genes revealed a region from -317 to -3 relative to the ATG, which is strongly conserved in both promoters. This 0.3 kb proximal promoter fragment is sufficient to direct flower-specific and MeJA-inducible GUS activity in transgenic potato plants, and thus contains a MeJA-responsive element that mediates induction by MeJA. Dimeric TGACG motifs or G-box elements similar to those found in other MeJA-inducible genes are not observed in this region, which suggests that a different DNA sequence is involved in MeJA induction of the LAP genes.

Permeabilized Arabidopsis protoplasts provide new insight into the chromatin structure of plant alcohol dehydrogenase genes.

New data from permeabilized protoplasts have expanded our view of the 5'DNase I hypersensitive area of the Arabidopsis Adh gene derived from nuclei. DNase I hypersensitivity analyses conducted with permeabilized protoplasts from Arabidopsis cell cultures indicates that there are four distinct sites of hypersensitivity centered around positions -425, -325, -200, and -60. The hypersensitive site at -200 coincides with an in vitro hypersensitive site created by purified transcription factors bound to a G-box element. The G-box is a functional cis element that plays a role in the signal transduction of hypoxia and other stresses in Adh. The data presented in this paper support the notion that G-box-related elements may also play a role in defining chromatin structure. The new Arabidopsis data are discussed within the context of what is known about the chromatin structures and regulation of two other plant Adh genes; maize Adh1 and Adh2. The chromatin of the maize Adh1 promoter is divided into a region that is constitutively hypersensitive to DNase I (-700 to -160) and an inducibly hypersensitive region (-140 and -40). There are several sequence elements within the hypersensitive regions bound by proteins in vivo. The anaerobic response element is the most well characterized and functions in the detection of hypoxia. The maize Adh2 gene promoter is constitutively hypersensitive to DNase I, with the exception of a small region that extends to include the TATA box as the gene becomes active. Several cis elements in the Adh2 promoter are bound by factors in vivo. One, at -160, is a functional element that acts as an activator in vascular tissue. The overall goal of our research with the Adh genes from maize and Arabidopsis is to gain further insight into the relationships between the regulation of gene transcription and chromatin structure in plants as it is clear that all the necessary components that characterize regulated gene activity may not be found simply by elucidating the linear sequence of nucleotides that lie 5' to the protein coding regions and finding proteins capable of binding the promoter in vitro.

Overexpression of deltaEmBP, a truncated dominant negative version of the wheat G-box binding protein EmBP-1, alters vegetative development in transgenic tobacco.

As a first step toward elucidating the in vivo function of plant bZIP proteins and their related G-box cis elements, we have introduced a dominant negative inhibitor of G-box-dependent transcriptional activation into tobacco plants by transforming them with a truncated EmBP-1 gene (deltaEmBP) containing the DNA binding and dimerization domains under the control of the CaMV 35S promoter. Five independent lines of transgenic plants expressing deltaEmBP were identified, as demonstrated by immunodetection of the transgenic protein in leaf extracts, and the ability of the protein to bind a target G-box DNA sequence. The transgenic plants exhibited an abnormal phenotype characterized by interveinal chlorosis, growth inhibition and weakening of stems and petioles, the severity of which positively correlated with deltaEmBP expression and G-box DNA binding capability. Furthermore, development of chlorosis and growth inhibition was dependent on growth irradiance. Low light promoted the development of interveinal chlorosis and growth inhibition in the transgenic plants, whereas high light conditions led to near-complete amelioration of the abnormal phenotype. Transgenic plants under both light regimes showed signs of impaired stem and petiole function which was not observed in wild-type tobacco. RhcS gene expression was not significantly altered by deltaEmBP expression, suggesting that down-regulation of this gene was not responsible for the altered phenotype. The results suggest that G-box elements specific for the EmBP-1 class of bZIP proteins have an important developmental function in vegetative plant tissues, and that the trans-dominant negative mutant approach is a useful tool for continued in vivo functional analysis of bZIP transcription factors and their corresponding cis elements in plants.

Effect of the nuclear factors EmBP1 and viviparous1 on the transcription of the Em gene in HeLa nuclear extracts.

Templates constructed from the wheat Em and maize rab28 promoters are efficiently and accurately transcribed in the well-characterized cell-free transcription system prepared from HeLa nuclei. Deletion analysis of the Em promoter indicates that a G-box (CACGTG) element (Em1b) is required for transcription. USF, a Myc transcription factor in HeLa nuclear extracts, activates transcription by binding to Em1b, as shown by the ability of an antibody raised against USF to inhibit transcription and to interfere with Em1b complex formation in an electrophoretic mobility shift assay. The addition of the recombinant Viviparous1 protein from maize to HeLa nuclear extracts specifically stimulated transcription of the Em promoter but was dependent on the presence of USF in the extract. In USF-depleted extracts, the addition of recombinant EmBP1, a basic leucine zipper transcription factor from wheat, activated transcription through Em1b as well as from a similar G-box in the adenovirus major late promoter. Our study demonstrates that the basic transcriptional apparatus in HeLa nuclear extract supports transcription from plant promoters and can be used to assay the function of certain plant nuclear proteins, thereby helping to determine their effects on transcription.

Improved ballistic transient transformation conditions for tomato fruit allow identification of organ-specific contributions of I-box and G-box to the RBCS2 promoter activity.

An improved protocol for the ballistic transient transformation of developing tomato (Lycopersicon esculentum) fruits is reported, which allows high-resolution cis-analysis of fruit-specific transcriptional activation. The tomato RBCS2 promoter fused to the firefly luciferase gene was used as a model system for this study. Osmotic treatment of fruit slices before, during and after particle bombardment, together with the optimization of bombardment conditions, resulted in a 100-fold increase in RBCS2 promoter-driven transient luciferase expression compared with previously reported protocols. Under these conditions, the transformed RBCS2 promoter was shown to be properly regulated in a developmental fashion. A cis-analysis of the RBCS2 promoter was performed. A 37 bp domain is required for high-level RBCS2 promoter activity both in leaves and young fruits. Two conserved sequence elements within this domain, an I-box element (GATAAG) and a G-box element (CACGTG), are necessary for its activity in leaves. In contrast, in young fruits, the G-box is the single dominant cis-acting element. These findings are discussed with respect to the proposed functions of G-box and I-box binding factors in regulating plant genes in different organs.

Rapid stimulation of a soybean protein-serine kinase that phosphorylates a novel bZIP DNA-binding protein, G/HBF-1, during the induction of early transcription-dependent defenses.

The G-box (CACGTG) and H-box (CCTACC) cis elements function in the activation of phenylpropanoid biosynthetic genes involved in the elaboration of lignin precursors, phytoalexins and the secondary signal salicylic acid as early responses to pathogen attack. We have isolated a soybean cDNA encoding a novel bZIP protein, G/HBF-1, which binds to both the G-box and adjacent H-box in the proximal region of the chalcone synthase chs15 promoter. While G/HBF-1 transcript and protein levels do not increase during the induction of phenylpropanoid biosynthetic genes, G/HBF-1 is phosphorylated rapidly in elicited soybean cells, almost exclusively on serine residues. Using recombinant G/HBF-1 as a substrate, we identified a cytosolic protein-serine kinase that is rapidly and transiently stimulated in cells elicited with either glutathione or an avirulent strain of the soybean pathogen Pseudomonas syringae pv. glycinea. Phosphorylation of G/HBF-1 in vitro enhances binding to the chs15 promoter and we conclude that stimulation of G/HBF-1 kinase activity and G/HBF-1 phosphorylation are terminal events in a signal pathway for activation of early transcription-dependent plant defense responses.

Transcription factor veracity: is GBF3 responsible for ABA-regulated expression of Arabidopsis Adh?

Assignment of particular transcription factors to specific roles in promoter elements can be problematic, especially in systems such as the G-box, where multiple factors of overlapping specificity exist. In the Arabidopsis alcohol dehydrogenase (Adh) promoter, the G-box regulates expression in response to cold and dehydration, presumably through the action of abscisic acid (ABA), and is bound by a nuclear protein complex in vivo during expression in cell cultures. In this report, we test the conventional wisdom of biochemical approaches used to identify DNA binding proteins and assess their specific interactions by using the G-box and a nearby half G-box element of the Arabidopsis Adh promoter as a model system. Typical in vitro assays demonstrated specific interaction of G-box factor 3 (GBF3) with both the G-box and the half G-box element. Dimethyl sulfate footprint analysis confirmed that the in vitro binding signature of GBF3 essentially matches the footprint signature detected in vivo at the G-box. Because RNA gel blot data indicated that GBF3 is itself induced by ABA, we might have concluded that GBF3 is indeed the GBF responsible in cell cultures for binding to the Adh G-box and is therefore responsible for ABA-regulated expression of Adh. Potential limitations of this conclusion are exposed by the fact that other GBFs bind the G-box with the same signature as GBF3, and subtle differences between in vivo and in vitro footprint signatures indicate that factors other than or in addition to GBF3 interact with the half G-box element.