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Plant Physiol. Aug 2004; 135(4): 2150–2161.
PMCID: PMC520786

Pathogen- and NaCl-Induced Expression of the SCaM-4 Promoter Is Mediated in Part by a GT-1 Box That Interacts with a GT-1-Like Transcription Factor1


The Ca2+-binding protein calmodulin mediates cellular Ca2+ signals in response to a wide array of stimuli in higher eukaryotes. Plants express numerous CaM isoforms. Transcription of one soybean (Glycine max) CaM isoform, SCaM-4, is dramatically induced within 30 min of pathogen or NaCl stresses. To characterize the cis-acting element(s) of this gene, we isolated an approximately 2-kb promoter sequence of the gene. Deletion analysis of the promoter revealed that a 130-bp region located between nucleotide positions −858 and −728 is required for the stressors to induce expression of SCaM-4. A hexameric DNA sequence within this region, GAAAAA (GT-1 cis-element), was identified as a core cis-acting element for the induction of the SCaM-4 gene. The GT-1 cis-element interacts with an Arabidopsis GT-1-like transcription factor, AtGT-3b, in vitro and in a yeast selection system. Transcription of AtGT-3b is also rapidly induced within 30 min after pathogen and NaCl treatment. These results suggest that an interaction between a GT-1 cis-element and a GT-1-like transcription factor plays a role in pathogen- and salt-induced SCaM-4 gene expression in both soybean and Arabidopsis.

Plant cells, like animal cells, elevate their cytosolic free-calcium levels ([Ca2+]cyt) with varying amplitude, frequency, and duration in response to a variety of external stimuli (Thomas et al., 1996; Berridge, 1997; McAinsh and Hetherington, 1998). The stimulus-specific [Ca2+]cyt transients are sensed by intracellular Ca2+-binding proteins, of which calmodulin (CaM) is one of the best characterized (Chin and Means, 2000; Snedden and Fromm, 2001). CaM is a ubiquitous intracellular mediator of Ca2+ signals having four helix-loop-helix Ca2+-binding motifs referred to as EF-hands (Babu et al., 1988). The Ca2+-bound CaM transduces the signals into many cellular processes through modulation of a variety of CaM-binding proteins, including enzymes such as kinases, phosphatases, and nitric-oxide synthase, as well as receptors, ion channels, G-proteins, and transcription factors (Liao et al., 1996; Snedden and Fromm, 1998; Lee et al., 1999a; Zuhlke et al., 1999).

In plant cells, in contrast to mammalian cells, multiple CaM genes code for a number of CaM isoforms. This has been shown in wheat (Triticum aestivum; Yang et al., 1996), potato (Solanum tuberosum; Takezawa et al., 1995; Poovaiah et al., 1996), and soybean (Glycine max; Lee et al., 1995a), among others. Over 30 genes encoding CaM isoforms are found in the Arabidopsis genome (The Arabidopsis Genome Initiative, 2000). We have recently cloned five CaM isoforms from soybean (SCaM-1-5). Although SCaM-1-3 are more than 90% identical to mammalian CaM, SCaM-4 and SCaM-5 exhibit only a 78% homology with SCaM-1 and are therefore the most divergent isoforms reported thus far in the plant and animal kingdoms. SCaM-4 is considered to be a bona fide CaM isoform based on the following characteristics. In its primary protein structure, SCaM-4 has four conserved putative EF-hands and a central linker region, hallmark structural features of CaM (Lee et al., 1995a). In addition, most of the nonconsensus amino acids occur outside the EF-hands, and the total number of amino acid residues is also conserved (Lee et al., 1995a). When compared with the consensus amino acid sequence of EF-hands derived from known Ca2+-binding proteins, the residues in all four Ca2+-binding loops of SCaM-4 conform to the consensus (Falke et al., 1994; Choi et al., 2002), suggesting that SCaM-4 can bind four Ca2+ molecules. Furthermore, SCaM-4 has the ability to modulate the activity of many CaM-dependent enzymes.

SCaM-4 can be distinguished from SCaM-1 by the target enzymes that it can activate (Lee et al., 1995a, 2000; Cho et al., 1998; Kondo et al., 1999; Chung et al., 2000). Some enzymes, including phosphodiesterase, plant Ca2+-ATPase, plant Glu decarboxylase, and CaM-dependent protein kinase II, can be activated equally well by either SCaM-1 or SCaM-4. However, other enzymes can only be activated by one isoform. For example, only SCaM-1 activates calcineurin, myosin light chain kinase, red blood cell Ca2+-ATPase, and plant NAD kinase, and only SCaM-4 activates nitric-oxide synthase. SCaM-1 and SCaM-4 also exhibit differences in the Ca2+ concentrations required for target enzyme activation (Lee et al., 2000).

All SCaM isoforms, including SCaM-4, are ubiquitously expressed in various plant tissues and show similar subcellular localization patterns to those of SCaM-1 (Lee et al., 1995a, 1999b). Intriguingly, the cellular level of SCaM-4 rapidly and dramatically rises in response to specific stimuli such as pathogens. Moreover, transgenic tobacco and Arabidopsis plants overexpressing SCaM-4 or SCaM-5 under the control of the cauliflower mosaic virus (CaMV) 35S promoter increase their resistance to pathogens by forming spontaneous hypersensitive response-like lesions with elevated expression of systemic acquired resistance-associated genes. This suggests that plant CaM isoforms have different physiological functions in vivo (Heo et al., 1999).

Although we know that the expression of CaM isoforms is differentially regulated by specific external stimuli, the cis- and trans-acting elements involved in plant CaM gene expression have not been well characterized. In this study, we have isolated and characterized the promoter sequence of the SCaM-4 gene. Core cis-acting elements that regulate expression of the SCaM-4 gene in response to pathogen infection or salt stress were identified within the SCaM-4 promoter between −1,215 and −1,150 bp, and between −858 and −728 bp. Here we report that an interaction between a GT-1 cis-element and a GT-1-like transcription factor plays a role in pathogen- and salt-induced SCaM-4 gene expression.


Isolation of the SCaM-4 Promoter and Analysis of Tissue-Specific Expression of the ScaM-4 Promoter-β-Glucuronidase Reporter Gene

To characterize the regulatory mechanisms controlling transcription of the SCaM-4 gene, we isolated its promoter region. Figure 1 shows the sequence of the SCaM-4 promoter (−1,286 bp to +765 bp), which extends into the 5′-untranslated region (GenBank accession no. AY052528). For comparative purposes, 2.4 kb of the 5′-upstream region of SCaM-1 was isolated from a soybean genomic library using SCaM-1 cDNA as a probe (GenBank accession no. AY052527; data not shown). We used a primer extension analysis to map the start site of SCaM-4 transcription. Two long extension products were detected 689 bp and 683 bp upstream of the first ATG site, suggesting heterogeneity in the mRNA 5′ ends or premature arrest of the reverse transcriptase (data not shown). The G residue corresponding to the longer extension product was taken to be the transcription start site and was numbered +1. As shown in Figure 1, a putative TATA box sequence is located upstream (nucleotides −33 to −37) of the transcription start site.

Figure 1.
Nucleotide sequence of the promoter region of the SCaM-4 gene. Sequences of the 5′-flanking region and the first exon of the SCaM-4 gene are shown together with a partial amino acid sequence from the 5′-end of the SCaM-4 coding region. ...

We then examined the tissue-specific expression pattern of an ScaM-4 promoter-β-glucuronidase (GUS) reporter in transgenic Arabidopsis to see whether it matched the expression pattern of ScaM-4 gene in soybean (Lee et al., 1995a, 1999b). Figure 2 shows representative examples of the tissue-specific expression of the SCaM-4 promoter-GUS gene. The SCaM-4 promoter-GUS was expressed primarily in the apical meristem (Fig. 2D) and hypocotyl regions of transgenic Arabidopsis seedlings (Fig. 2, C and F). The GUS staining patterns in transgenic Arabidopsis seedlings were similar to the expression patterns of SCaM-4 mRNA and SCaM-4 protein in soybean seedlings (Lee et al., 1995a, 1999b).

Figure 2.
Histochemical localization of the expression of GUS fused to the SCaM-4 2-kb promoter in transgenic Arabidopsis seedlings. A, Structure of the pBI 4D1, a binary vector used for SCaM-4 promoter-GUS expression. The bacterial neomycin phosphotransferase ...

Analysis of the Effect of Signaling Molecules on Expression of the SCaM-4 Promoter-GUS Reporter Gene

Expression of plant CaM genes has been shown to respond to various environmental stresses including light, phytohormones, touch, wounding, high salinity, and pathogens (Jena et al., 1989; Braam and Davis, 1990; Botella and Arteca, 1994; Harding et al., 1997). The expression of the two soybean CaM genes encoding the SCaM-1 and SCaM-4 isoforms was examined after treatment of soybean suspension-culture cells (W82) with a soybean pathogen, Pseudomonas syringae pv glycinea A (Psg), or with 150 mm NaCl (Fig. 3A). SCaM-4 mRNA levels peaked at 0.5 h following pathogen or NaCl treatment and then slowly declined to basal levels by 12 h. In contrast, the expression of SCaM-1 was not activated by the same treatments (Fig. 3A). Similarly, 4-week-old transgenic Arabidopsis plants carrying the SCaM-4 2-kb promoter-GUS reporter were treated with a pathogen, P. syringae pv tomato DC3000 (PsD), or with 150 mm NaCl to examine the expression pattern of the SCaM-4 promoter in a heterologous system (Fig. 3B). Gel blots of total RNA isolated from the transgenic Arabidopsis plants were probed with GUS cDNA. GUS mRNA levels appeared at high levels by 0.5 h after application of the pathogen or NaCl, but returned to nearly basal levels by 24 h, despite the continued presence of the stressor (Fig. 3B). In addition, the expression of the SCaM-4 gene in soybean suspension-culture cells (W82) was dramatically induced within 1 h after treatment with Psg, glycol chitin, NaCl, or Ca2+-ionophore A23187 (Fig. 3C). The application of exogenous KCl, mannitol, hydrogen peroxide (H2O2), salicylic acid, jasmonic acid, or abscisic acid (ABA) did not induce the expression of SCaM-4 (Fig. 3C).

Figure 3.
Expression pattern of the SCaM-4 and SCaM-1 genes in soybean and GUS reporter expression driven by the SCaM-4 promoter in response to various treatments. A, Time-course of accumulation of SCaM-4 and SCaM-1 transcripts after pathogen or NaCl treatment ...

We then examined the effects of these treatments on GUS reporter gene expression in Arabidopsis leaf protoplasts, a plant transient expression system. After treatment with various biotic and abiotic signals, we determined the level of induction of GUS activity with reference to luciferase (LUC) activity. The GUS activity of the SCaM-4 promoter-GUS reporter was enhanced about 3- to 7-fold when treated with PsD, glycol chitin, NaCl, or Ca2+-ionophore A23187 (Fig. 3D). However, the other treatments did not increase GUS activity. We also examined the effects of the treatments on the expression of 2.4-kb SCaM-1 promoter-GUS construct. No GUS induction was observed for the SCaM-1 promoter in Arabidopsis protoplast cultures, similar to the expression pattern found in soybean seedlings (Fig. 3E). Overall, these experiments show that Arabidopsis can be a useful system in which to study the pathogen and NaCl responsive regulatory elements of the ScaM-4 promoter.

Deletion Analysis of the SCaM-4 Promoter

To determine the specific regions of the promoter that are involved in SCaM-4 induction by pathogen or NaCl treatments, a series of 5′ deletions were made in the SCaM-4 promoter region (Fig. 4A). Each construct was transiently introduced into Arabidopsis protoplasts by polyethylene glycol-mediated transformation, and GUS activity was assayed after treatment with 150 mm NaCl, or PsD for 12 h. The GUS reporter gene was strongly induced by pathogen or NaCl in constructs containing deletions up to −1,286 (pBI 4D1) or −858 (pBI 4D2), but this induction was completely lost in the construct containing a deletion up to −566 (pBI 4D3). Furthermore, the pBI 4delA construct, containing nucleotides −1,286 to −728, showed maximal GUS induction after treatment with pathogen or NaCl (about 14- and 19-fold, respectively), a pattern of induction very similar to that of the SCaM-4 2-kb promoter (Fig. 4B).

Figure 4.
Quantitative fluorometric assays for GUS activity driven by various SCaM-4 promoter deletion constructs. A, Diagram of various deletion derivatives of the SCaM-4 promoter. Deletion end points are indicated in bp from the transcription start site (indicated ...

To determine the region(s) within the −1,286 to −728 bp region that are responsible for induction by pathogen and NaCl treatments, the SCaM-4 promoter region was further divided into six overlapping fragments of 100 to 200 bp in length, and the fragments were fused to the upstream region of the TATA minimal promoter contained within the pDel. 151-8 vector (Sundaresan et al., 1995; Fig. 4C). These constructs were then tested in transient expression assays in Arabidopsis protoplasts treated for 12 h with water (control), pathogen, or NaCl (Fig. 4D). The −1,286 to −728 construct (pBI 4delA) showed GUS induction of about 8- and 11-fold after treatment with pathogen and NaCl, respectively. The constructs containing the regions −1,286 to −1,065 (pBI 4delB), −858 to −728 (pBI 4delD), −1,065 to −728 (pBI 4delE), and −1,286 to −858 (pBI 4delG) showed GUS activities that were approximately one-half of those of the pBI 4delA construct. In the construct containing the regions −1,286 to −1,065 and −858 to −728 (pBI 4delF), the GUS activity was increased about 6- and 9-fold after treatment with pathogen and NaCl, respectively.

Pathogen and NaCl-Induced Expression of SCaM-4 Promoter-GUS Involves a GT-1 cis-Acting Regulatory Element

From GUS assays in Arabidopsis protoplasts containing the deletion constructs of the SCaM-4 promoter in vivo, we identified nucleotides −1,286 to −1,065 and −858 to −728 as important elements for pathogen and NaCl responses. To test whether these regions interact specifically with nuclear proteins, we divided the 1.3-kb promoter region into five fragments (fragments A–E), as shown in Figure 5A. Each of the five double-stranded fragments was used in an initial series of electrophoretic mobility shift assays (EMSAs) with soybean nuclear extracts from W82 cells treated for 1 h with 10 mm MgCl2 (control), pathogen (Psg), or NaCl. Fragments containing the −1,286 to −1,065 (A) and −858 to −549 (C) regions each gave one major retarded band when incubated with Psg- or NaCl-treated nuclear extracts (Fig. 5A). Fragment C (−858 to −549) was further subdivided into three overlapping fragments, C-1, C-2, and C-3, and each was used in EMSAs with the nuclear extracts described above. Only the C-1 region fragment (−858 to −728) showed a strong mobility shift when incubated with Psg-treated nuclear extracts (Fig. 5B). The mobility shift was completely blocked by the addition of a 50-fold molar excess of unlabeled C-1 but not by an excess of unlabeled C-2 or C-3. EMSAs using the C-1 region as a probe with heat-treated (65°C, 5 min) or proteinase K-treated nuclear extracts, showed that the DNA-binding complex of the C-1 region was heat stable but sensitive to proteinase K digestion (data not shown).

Figure 5.
Identification of a GT-1 cis-element involved in SCaM-4 gene expression in response to pathogen or NaCl. The binding reaction mixture of each experiment (20 μL) contained 32P-labeled DNA probe (40 kcpm), poly[dI/dC] (2 μg), and nuclear ...

To more precisely define the position of the protein binding site, we designed nine double-stranded oligonucleotides of 15 to 30 bp in length (E1–E9, Fig. 5C), which were used as C-1 competitors in EMSAs. The E4 oligonucleotide completely blocked nuclear protein binding to the C-1 fragment (Fig. 5D). Examination of the sequence of the E4 fragment revealed that it contains a GT-1 cis-element (GAAAAA). DNase I footprinting assays using the C-1 fragment and pathogen-treated nuclear extracts confirmed that the GT-1 element is indeed recognized by nuclear factors (data not shown). The involvement of the GT-1 cis-element in binding to pathogen-treated nuclear extracts was tested using a subset of oligonucleotides derived from E4 fragments (Fig. 5E). While E4-1 (TAAGAAAAATAA) effectively bound to pathogen treated nuclear extracts, the mutations E4-1(M1; TAACAAAAATAA) and E4-1(M2; TAACCAAAATAA) caused significant reductions in protein binding (Fig. 5E).

To examine whether the GT-1 cis-element of the SCaM-4 promoter, identified by in vitro DNA binding, actually plays a role in the cellular responses to pathogen and NaCl-induction, we generated a mutant ScaM-4 promoter (−1,286 to −728)-GUS construct that contains a GA to CC mutation in the GT-1 element (Fig. 6A). The pBI 4delA (−1,286 to −728) showed a 7- to 8-fold induction of the GUS reporter gene after treatment with NaCl or pathogen. However, the pBI 4delA M2 mutant construct repeatedly showed 4- to 5-fold induction by the same treatment, approximately a 30% reduction compared to the wild type promoter (Fig. 6B). This result shows that while the GT-1 element is involved in the expression of ScaM-4, the −1,065 to −1,286 region also plays a role in the NaCl- and pathogen-induced expression of the SCaM-4 gene (Fig. 5A).

Figure 6.
Effect of mutation of the GT-1 cis-element within the SCaM-4 promoter on expression of the SCaM-4 2 kb promoter-GUS reporter gene in Arabidopsis protoplasts. A, Schematic diagram of a SCaM-4 promoter construct of the −1,286 to −728 bp ...

Isolation of a Transcription Factor Interacting with the SCaM-4 GT-1 cis-Element

As a first approach to isolate the transcription factor that interacts with the GT-1 cis-element within the SCaM-4 promoter, we searched the complete genome of Arabidopsis. Seventeen sequences encoding trihelix DNA-binding factors (or GT transcription factors) have been found in the Arabidopsis genome (Ayadi et al., 2004). The analysis identified four GT-1 related transcription factors with a single trihelix motif: AtGT-1 (At1g13450), AtGT-4 (At3g25990), AtGT-3a (At5g01380), and AtGT-3b (At2g38250). AtGT-3a and AtGT-3b showed low homology to AtGT-1 (less than 36% sequence identity). However, both these factors contain a conserved trihelix DNA-binding domain that is also found in the N-terminal region of AtGT-1 (Fig. 7A).

Figure 7.
Alignment of the deduced amino acid sequence and northern-blot analysis of GT-1 related transcription factor genes isolated from Arabidopsis. A, Alignment of the deduced amino acid sequences of four Arabidopsis genes encoding GT-1 related factors; AtGT-1 ...

The full-length cDNA clones for the three AtGT-1-related proteins (AtGT-1, AtGT-4, and AtGT-3b) were isolated by the reverse transcription (RT)-PCR method. The expression patterns of the three AtGT-1-related genes were examined in pathogen- and NaCl-treated Arabidopsis plants by northern-blot analysis. As shown in Figure 7B, treatment of the plants with pathogen or NaCl resulted in a rapid increase in the transcription of AtGT-3b. However, the expression levels of the two other AtGT-1-related transcription factors were not changed by treatment with pathogen or NaCl. Therefore, AtGT-3b was selected as a good candidate for a transcription factor which binds to the GT-1 cis-element of the SCaM-4 promoter during the plant response to pathogen attack and NaCl stress.

The AtGT-3b gene has an open reading frame of 870 bp, which would encode a protein of 289 amino acids with a molecular mass of approximately 31.8 kD (Fig. 7A). The deduced amino acid sequence of AtGT-3b contains two different nuclear localization signal sequences, one of which corresponds to a bipartite-type nuclear localization signal within a trihelix domain (KRNKLLWEVISNKMRDK) located between amino acids 65 and 81. The other corresponds to a simian virus 40 (SV 40)-type nuclear localization signal located in the C-terminal region (KKRK) encompassing amino acids 185 to 188.

The AtGT-3b protein was further analyzed for interactions with the GT-1 cis-element in the SCaM-4 promoter in vitro and in a yeast selection system. To test the binding activity of AtGT-3b to the GT-1 cis-element within the E4 fragment of the SCaM-4 promoter, we produced a recombinant AtGT-3b protein fused to glutathione S-transferase (GST) in Escherichia coli. As shown in Figure 8A, the ability of the recombinant GST-AtGT-3b fusion protein to bind to the E4 oligonucleotide was validated by EMSA. The DNA binding specificity of GST-AtGT-3b was also confirmed by competition experiments (Fig. 8A). A 200-fold molar excess of unlabeled E4 oligonucleotide completely blocked E4 binding to GST-AtGT-3b (Fig. 8A, lane 9). In contrast, neither AtGT-1 nor AtGT-4 formed protein-DNA complexes under these conditions (data not shown).

Figure 8.
Interaction of AtGT-3b with the GT-1 cis-element within the E4 fragment of the SCaM-4 promoter in vitro and in a yeast selection system. A, EMSA using bacterially produced recombinant AtGT-3b protein and E4 oligonucleotide. The 32P-labeled E4 oligonucleotide ...

The interaction of the AtGT-3b protein with the GT-1 cis-element was reconfirmed with a yeast selection system. We constructed a YM4271 yeast strain carrying integrated copies of HIS3 and lacZ as dual reporter genes with four tandem repeats of the E4 fragment upstream of the TATA element. The yeast cells were then transformed with AtGT-3b cDNA fused to the transcriptional activation domain of yeast GAL4 (Fig. 8B). As predicted by the in vitro binding assay, the AtGT-3b protein and the E4 fragment conferred HIS selection in the presence of 45 mm 3-AT, a competitive inhibitor of the HIS3 gene product (HIS3p). This result provides strong evidence for an interaction in the yeast system. In contrast, yeast cells carrying plasmids with cDNA inserts of AtGT-1 or AtGT-4 did not grow on medium lacking His in the presence of 45 mm 3-AT (data not shown).


The expression of plant CaM and CaM-like genes from a number of species is differentially regulated in response to external stimuli of both abiotic (e.g. light, gravity, heat, touch, cold, salinity, and drought) and biotic (e.g. phytohormones and pathogens) origins (Snedden and Fromm, 1998, 2001). We have previously shown that SCaM-4 and SCaM-5 are more rapidly induced by fungal elicitors or pathogens than are the SCaM-1, -2, and -3 isoforms (Heo et al., 1999). Here we have shown that the mRNA of SCaM-4 is also significantly induced in response to salt stress (Fig. 3A). Furthermore, our group has already shown that SCaM-4 activates a different pattern of CaM-dependent enzymes than SCaM-1 (Lee et al., 2000). These findings suggest that particular Ca2+/CaM signaling pathways can be mediated by different CaM isoforms, which, in turn, may give plant cells the ability to have diverse cellular responses to Ca2+ signals. To elucidate the differences between SCaM-1 and SCaM-4 at the level of transcriptional regulation, we isolated the 5′-flanking regions of the SCaM-1 gene (2.4 kb) and the SCaM-4 gene (2 kb).

To further understand the upstream signaling mechanisms of SCaM-4 gene expression in response to pathogen and NaCl signals, we analyzed the cis- and trans-acting elements involved in SCaM-4 gene expression. Based on a report that Arabidopsis also contains SCaM-4/-5 gene homologs (Zielinski, 2002), we prepared transgenic Arabidopsis carrying soybean SCaM-4 2-kb promoter-GUS constructs and found that GUS was expressed in the apical meristem and hypocotyl regions but not in root tissues. This is consistent with the results of a northern-blot analysis of soybean seedlings (Lee et al., 1995a). This result strongly suggests that the expression patterns of the SCaM-4 promoter are maintained in heterologous transgenic Arabidopsis plants. The GUS activity of the SCaM-4 promoter was enhanced about 3- to 7-fold when treated with PsD, glycol chitin, NaCl or the Ca2+-ionophore A23187 (Fig. 3D), strongly suggesting that the SCaM-4 promoter responds to transcriptional regulators under various environmental stress conditions.

Using EMSAs, we examined protein-DNA interactions with the SCaM-4 promoter and the soybean nuclear extracts and precisely identified the cis-acting elements involved in plant responses to pathogen attack and NaCl stress. Two promoter regions, −1,286 to −1,065 (A) and −858 to −566 (C), were critical for the SCaM-4 promoter binding of nuclear extracts prepared from pathogen- or NaCl-treated soybean suspension culture cells (W82). This result is in good agreement with the data obtained from the in vivo transient expression assay using Arabidopsis protoplasts. From a DNase I footprinting analysis and EMSA using synthetic oligonucleotides, we identified a GT-1 cis-element within a subfragment of the C region, the E4 fragment, as an important element involved in SCaM-4 gene expression (Figs. 5 and and6).6). Additionally, a base substitution analysis demonstrated that GA in the GT-1 cis-element (5′-GAAAAA-3′) is required for binding to nuclear factor(s) in response to pathogen- or salt-induced stress. Together, these data imply that a GT-1-related transcription factor positively regulates SCaM-4 gene expression under the conditions of pathogen attack or NaCl stress.

The GT-1 cis-element, one of many cis-acting DNA elements found in plants, was first identified in pea (Pisum sativum) as the Box II element (5′GTGTGGTTAATATG3′) in the promoter of the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit gene (RBCS-3A; Green et al., 1987). Depending on the promoter structures, GT-1 cis-elements can have a positive or a negative effect on transcription. One common feature found in all GT-1 cis-elements is a core sequence of four or five nucleotides, which consists of T or A preceded by one or two G nucleotides at the 5′ end. The deduced consensus core sequence is currently defined as 5′-G-Pu-(T/A)-A-A-(T/A; Zhou, 1999). It is thought that the high degeneracy of the GT-1 cis-element partly explains its diverse functions as well as its light-specific regulatory functions.

In vitro experiments have shown that the GT-1 transcription factor can interact with the TFIIA-TBP-TATA complex, suggesting that GT-1 may activate transcription through direct interactions with the minimal preinitiation complex (Le Gourrierec et al., 1999). It has also been reported that GT-1 interacts with important enhancer regions in the Cpr (NADPH; cytochrome P450 reductase; Lopes Cardoso et al., 1997) and Str (Pasquali et al., 1999) genes, which are induced by fungal elicitors and yeast extracts, respectively. In this research, we showed that the AtGT-3b, a GT-1-like transcription factor, was rapidly induced by pathogen and salt stress. In addition, the AtGT-3b protein specifically bound to the GT-1 cis-element within the E4 fragment of the SCaM-4 promoter both in vitro and in a yeast selection system (Fig. 8). The induction by pathogen and NaCl stress along with specific binding to the GT-1 cis-element, both in the EMSAs and in the yeast selection system, strongly suggests that AtGT-3b is a transcription factor involved in SCaM-4 gene expression. Our results using soybean suspension-culture cells demonstrate that an AtGT-3b homolog might also exist in soybean plants, where it may mediate the induction of the SCaM-4 gene in response to pathogen or salt stress.

The data obtained from this study lead to a model in which environmental stresses induce SCaM-4 gene expression by mediating the binding of a GT-1-like transcription factor to the GT-1 cis-element (GAAAAA) within the −858 to −728 region of the SCaM-4 promoter. Additional binding event(s), mediated by yet to be defined trans-acting factor(s), may be required at the upstream −1,215 to −1,065 bp cis-element. Currently, we are characterizing the −1,286 to −1,065 bp region with respect to its contribution to the induction of SCaM-4 gene transcription in response to pathogens and NaCl. Interestingly, we have found a 65 bp sequence within the −1,215 to −1,150 region that is retarded in EMSAs from pathogen- or NaCl-treated nuclear extracts (data not shown). Further investigation into the regulation of SCaM-4 will involve characterization of other cis-elements and their cognate transcription factors. This will provide a better understanding of the roles played by DNA-protein interactions in SCaM-4 gene expression during plant defense responses.


Plant Materials and Bacterial and Yeast Strains

Soybean (Glycine max) cells (W82) were grown in suspension culture in Murashige and Skoog medium supplemented with 0.75 mg L−1 benzyl adenine, maintained at 25°C in the dark, and stirred at 130 rpm. Arabidopsis (ecotype Columbia) plants were used for the preparation of transgenic plants. For DNA cloning, Escherichia coli XL1-Blue MRF′ and DH 5α (Stratagene, La Jolla, CA) were used as bacterial strains. The expression of the GST-fusion protein was performed in E. coli, BL21 (pLys S) DE3. The yeast strain YM4271 (MATa, ura3-52, his3-200, ade2-101, lys2-801, leu2-3, 112, trp1-901, tyr1-501, gal4512, gal80538, ade5::hisG) was used for reporter vector integration in the yeast selection system (Wilson et al., 1991; Liu et al., 1993).

Isolation of the 5′ Upstream Sequences of SCaM-4 and SCaM-1

The 5′ upstream region of the SCaM-4 gene was obtained using the Universal Genome Walker kit (CLONTECH, Palo Alto, CA). First, separate aliquots of soybean genomic DNA were digested with five blunt-end restriction enzymes (EcoRV, ScaI, DraI, PvuII, and StuI), and ligated to Genome Walker adaptors. Primary PCR was performed using adaptor primer 1 (AP 1) and a SCaM-4 cDNA specific primer (5′-GTCCTCGGTAAGAAACAGACTCATCC-3′). The second PCR was performed using adaptor primer 2 (AP 2) and the same SCaM-4 cDNA specific primer. The amplified PCR products were examined on an agarose gel, and subcloned into the pGEM T-Easy vector. After sequencing of overlapping deletion products using the Erase-A-Base kit (Promega, Madison, WI), the 5′ upstream region of the 2-kb SCaM-4 gene was connected by asymmetric PCR.

The upstream region of the SCaM-1 gene was isolated by screening a soybean (Glycine max cv Williams 82) genomic DNA library constructed in bacteriophage λ Fix II (Stratagene, Heidelberg, Germany). A 2.4-kb internal EcoRI fragment that hybridized to the SCaM-1 cDNA probe was subcloned into the multiple cloning site of the pBluescript II SK (−) vector (Stratagene, La Jolla, CA). A sequential series of overlapping deletions from both ends were made using the Erase-A-Base kit (Promega) according to the manufacturer's protocol and sequenced.

RNA Gel-Blot Analysis

Various tissues of transgenic Arabidopsis plants and W82 cells collected on filter papers (Whatman, Clifton, NJ) by vacuum filtration were used for isolation of total RNA as described (Park et al., 2002). RNA gel-blot analyses were carried out as described previously (Sambrook et al., 1989). Gene-specific probes were made from the 3′-untranslated regions of each cDNA, using a 276-bp HaeIII/XhoI fragment of SCaM-1 and a 347-bp EcoRI/XhoI fragment of SCaM-4 cDNA. The 32P-labeled 1.87-kb GUS cDNA and three GT-1–related cDNA clones of Arabidopsis were used for hybridization.

Construction of Promoter Deletion Derivatives of the SCaM-4 Genes Fused to a GUS

For promoter analysis in transgenic plants and Arabidopsis protoplasts, SCaM-4 promoter-GUS-NOS cassette constructs were used. Deleted promoters were cloned into the SalI/BamHI sites of the binary vector pBI 101 (CLONTECH). The following deletion derivatives were cloned into the SalI/BamHI site of the binary vector, pBI 101: a SCaM-1 promoter containing a fragment from −2,230 to +84, named pBI 1D1, and a SCaM-4 promoter containing various fragments (−1,286 to +750, pBI 4D1; −858 to +750, pBI 4D2; −566 to +750, pBI 4D3; −217 to +750, pBI 4D4; and +34 to +750, pBI 4D5). For characterization of the promoter in more detail, the −1,286 to −728 bp region of the SCaM-4 promoter was subdivided into six different fragments and ligated into the region upstream of the TATA-containing minimal promoter of the pDel. 151-8 vector (Sundaresan et al., 1995). These fragments were as follows: −1,286 to −728, named pBI 4delA; −1,286 to −1,065, pBI 4delB; −1,065 to −858, pBI 4delC; −858 to −728, pBI 4delD; −1,065 to −728, pBI 4delE; −1,286 to −1,065, and −858 to −728, pBI 4delF; and −1,286 to −858, pBI 4delG. The SCaM-1/-4 promoter-GUS and SCaM-4 promoter deletion-GUS constructs were propagated in E. coli, XL1-Blue MRF′ (Stratagene). The plasmid constructs were isolated using CsCl gradients (Ausubel et al., 1987). The structures of all constructs were confirmed by sequencing or restriction digest mapping.

Arabidopsis Protoplast Transfection and Fluorometric GUS Assays

Isolation of Arabidopsis protoplasts and polyethylene glycol-mediated DNA transfection were performed as described previously (Abel and Theologis, 1994). Typically, 5 mL of Arabidopsis protoplast suspension (5 × 106 per mL) was cotransfected with 15 μg of a test construct and 5 μg of a CaMV 35S promoter-LUC control vector, pJD 300. The transfected Arabidopsis protoplasts were incubated in W5 solution under various conditions for 12 h in the dark at room temperature. Using the transfected protoplasts, GUS assays were performed fluorometrically with the substrate 4-methyl umbelliferyl glucuronide as described (Jefferson et al., 1987). LUC assays were performed using the Promega LUC assay system according to the manufacturer's instructions. In order to normalize for transfection efficiency, the CaMV 35S promoter-LUC plasmid was cotransfected in each experiment.

Plant Transformation and Histochemical GUS Assays

To generate transgenic Arabidopsis plants (ecotype Columbia), pBI 4D1 and pBI 101 plasmids were introduced into Agrobacterium tumefaciens GV3101 by electroporation, and Arabidopsis plants were transformed by vacuum infiltration (Clough and Bent, 1998). Histochemical GUS staining of transgenic Arabidopsis plants was performed according to a previously described method (Lee et al., 1995b).

Pathogen and Various Chemical Treatments

Different pathogenic bacteria (108 cfu/mL) were used for infection of the two plant species. Pseudomonas syringae pv glycinea carrying avrA (Psg) was used for infection of soybean suspension culture cells (W82), and P. syringae pv tomato DC3000 (PsD) was used for Arabidopsis plants. Bacteria grown in liquid King's medium were washed and resuspended in 10 mm MgCl2 (King et al., 1954). For northern-blot and GUS fluorometric assays, W82 cells and Arabidopsis protoplasts were treated with 0.05% glycol chitin, 150 mm KCl, 300 mm mannitol, 150 mm NaCl, Ca2++A23187 (25 μm Ca2+ ionophore A23187 plus 5 mm CaCl2), 2 mm hydrogen peroxide (H2O2), 2 mm salicylic acid, 100 μm jasmonic acid, or 100 μm ABA.

Preparation of Soybean Nuclear Extracts and Electrophoretic Mobility Shift Assays

Nuclear extracts were prepared from W82 cells that had been treated with MgCl2 (mock inoculation), pathogen, or 150 mm NaCl for about 1 h using a procedure described previously (Nagao et al., 1993). EMSAs were performed as described (Hong et al., 1995) using [32P]-labeled double-stranded DNA probes. The assay mixtures contained soybean nuclear extracts (4 μg of protein) or E. coli extracts (10 μg of protein), 4 × 104 cpm of each binding probe, 2 μg of poly[dI/dC], 20 mm HEPES-KOH (pH 7.9), 0.5 mm DTT, 0.1 mm EDTA, 50 mm KCl, and 15% glycerol in a 20 μL reaction volume. The mixtures were incubated at room temperature for 15 min and electrophoresed on 5% or 8% polyacrylamide gels in 0.5× TBE buffer. Subsequently, the gels were dried and exposed to x-ray films.


Total RNA was extracted from the pathogen- or 150 mm NaCl-treated samples of 4-week-old Arabidopsis seedlings. Total RNA (5 μg) was reverse-transcribed in a 50-μL reaction volume with 10 ng of oligo(dT)17 primer using Superscript RTase according to the manufacturer's protocols (BRL Life Technologies, Grand Island, NY). The following oligonucleotides were synthesized for amplification of GT-1–related cDNAs in Arabidopsis (Ayadi et al., 2004): AtGT-1 (At1g13450; upstream primer: 5′-GCGTCGACAATGTTCATTTCCGACAAATCTCGT-3′, downstream primer: 5′-CCGCTCGAGTCATCTCACACCTCGATACACAGC-3′), AtGT-3b (At2g38250; upstream primer: 5′-CGCGGATCCATGGATGGACATCAGCATCATCAC-3′, downstream primer: 5′-CCGCTCGAGTTAGAGGGAACCATCTCTAGTAAG-3′), and AtGT-4 (At3g25990; upstream primer: 5′-CGCGGATCCATGTTTGTTTCCGATA ACAACAAT-3′, downstream primer: 5′-CCGCTCGAGTCATCTCATTCCTCTGTA TAAGCG-3′). After a standard PCR of 30 cycles, aliquots were run on an agarose gel. Each fragment of accurate size was cloned into a pGEM-T Easy vector (Promega) and identified by DNA sequencing.

Expression of AtGT-3b in E. coli and Yeast Selection Analysis

An AtGT-3b cDNA fragment was prepared by PCR and cloned into the BamHI and XhoI sites of the pGEX-2T-linker I vector (Amersham Pharmacia Biotech, Uppsala). Using E. coli strain BL21 (pLys S) DE3, GST::AtGT-3b was overexpressed, and the bacterial supernatant was used for gel mobility shift assays. For analysis in the yeast selection system, construction of reporter plasmids and selection of the yeast reporter strain were performed according to the manufacturer's protocol (CLONTECH). To generate the AD-fused AtGT-3b cDNA construct, a BamHI/PstI fragment of AtGT-3b cDNA was ligated into the pGAD424 vector. Positive interactions were verified by judging yeast growth on SD medium containing 45 mm 3-AT and assaying for β-galactosidase.

Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY052528 for SCaM-1 promoter and AY052527 for SCaM-4 promoter.


We thank Dr. C. Lamb for providing the soybean suspension-culture cells, W82.


1This work was supported by a Basic Research Grant (grant no. R02–2002–000–00009–0), by the National Research Laboratory program (2000–N–NL–01–C–236), by the Crop Functional Genomics Center of the 21st Century Frontier Research Program CG1512, by the Gyeongnam High Tech Foundation (2001), and by the Ministry of Agriculture and Forestry (grant no. 298049–4 to M.J.C.), and partially by the Environmental Biotechnology Research Center (grant no. R15–2003–012–02003–0), by the Crop Functional Genomics Center of the 21st Century Frontier Research Program CG1124, and by the Center for Plant Molecular and Genetic Breeding Research, KOSEF in Korea (grant to J.C.H.). Y.H.K., C.Y.P., and B.C.M. were supported by scholarships from the BK21 program, Ministry of Education and Human Resources Development in Korea.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.041442.


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