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Original
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Acta Biochim Biophys
Sin 2006, 38: 46-52 |
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doi:10.1111/j.1745-7270.2006.00126.x |
Roles of Salicylic
Acid-responsive Cis-acting Elements and W-boxes in Salicylic Acid
Induction of VCH3 Promoter in Transgenic Tobaccos
Hai-Yan LI1,2,
Wei WEI2&, and Yu LI1*
1 Institute
of Mycology, Jilin Agricultural University, Changchun 130118, China;
2 College
of Biotechnology, Jilin Agricultural University, Changchun 130118, China
Received: July 25, 2005
Accepted: September 19, 2005
This work was supported
by the grants from the National Natural Science Foundation of China (30400300),
the Fund for Distinguished Young Scholars of Jilin Province and the Science and
Technology Development Projects of Jilin Province (20050117, 20030553-1 and
20040209-2)
&Present address:
Department of Biology, Graduate School of the Chinese Academy of Science,
Beijing 100039, China
*Corresponding
author: Tel, 86-431-4510966; Fax, 86-431-4510971; E-mail, [email protected]
Abstract������� A salicylic acid (SA)-inducible VCH3
promoter was recently identified from grapevine (Vitis amurensis) that
contains two inverse SA-responsive cis-acting elements and four W-boxes.
To further demonstrate� the roles of these elements, four fragments with
lengths from -1187, -892,
-589, -276
to +7 bp were fused with the b-glucuronidase (GUS) reporter
gene and transferred to Nicotiana tobacum, together with another four VCH3
promoter fragments with mutation in the two inverse SA-responsive elements. The
functions of each promoter fragment were examined by analysis of GUS activity
in the transgenic tobacco root treated with SA. Enhanced GUS activity was shown
in the roots of transgenic tobaccos with the VCH3 (-1187)-GUS construct containing two
SA-responsive cis-acting elements and four W-boxes. However, GUS
activity directed by the VCH3 (-892)-GUS
construct, containing one SA cis-acting element and four W-boxes, was
reduced by up to 35% compared
with that in tobaccos transformed with the VCH3 (-1187)-GUS construct, indicating that the SA cis-acting
element plays an important role in SA induction of the VCH3 promoter.
Neither the m2VCH3 (-1187)-GUS nor the mVCH3
(-892)-GUS construct,
with mutation on the SA-responsive elements, abolished the expression of GUS
activity, demonstrating that
the W-boxes in the VCH3 promoter are also involved in SA
induction. Histochemical analysis of GUS activity directed by each of the eight
VCH3 promoter fragments showed that GUS was expressed specifically in
vascular tissue. It was concluded that both the SA-responsive cis-acting
elements and the W-boxes are important for the SA induction of the VCH3
promoter. This promoter might have a potential use in plant genetic
engineering.
Key words������� VCH3 promoter; salicylic acid (SA)-responsive
cis-acting element; W-box; site-directed mutagenesis; SA induction;
transgenic tobacco
Salicylic acid (SA) is one of the important signal molecules involved in disease resistance in plants [1,2]. An increased level of SA is required to activate the transcription of defense genes and to develop an efficient pathogen resistance response [3]. In addition, the accumulation of SA and the activation of defense genes, such as pathogenesis-related (PR) genes, have been reported to occur after exposure of plants to ozone, ultraviolet radiation, cold or high salinity [4-6]. Therefore, it is considered that SA plays a crucial role under either biotic or abiotic stress, and that SA-inducible promoters might have an important use in engineering stress-tolerant plants to drive gene expressions when necessary.
To date, many plant gene promoters induced by SA have been reported, such as soybean IFS promoter, tobacco PR-1a and PR-2d promoters, Gastrodia elata GAFP-2 promoter and Arabidopsis GST6 promoter [7-11]. Of these promoters, the SA-responsive cis-acting element TGACG, which belongs to the family of activation sequence-1 elements, is reported to function as a transcriptional enhancer conferring SA inducibility to reporter genes in transgenic plants [8-10]. The W-box (T)TGAC(C/T), another cis-acting DNA element found frequently in the promoter of defense-related genes [12-14], is recognized specifically by pathogen- or SA-induced WRKY DNA binding protein [15-17]. Recently, Rocher et al. reported the W-box was required for full expression of the SA-responsive gene SFR2 [18]. We previously isolated a 1216 bp VCH3 promoter from grapevine (Vitis amurensis), which contains two inverse SA-responsive cis-acting elements and four W-boxes, and is strongly induced by SA.
In the present work, we performed deletion analysis and site-specific mutagenesis to further demonstrate the roles of these promoter elements in response to SA induction in the VCH3 promoter. The tissue-specific expression patterns of the b-glucuronidase (GUS) reporter gene directed by the wild-type and mutant VCH3 promoter fragments are also discussed.
Materials and Methods
Materials
Escherichia coli strain DH5a, Agrobacterium tumefaciens� strain LBA4404, binary vector pBI121 and tobacco (Nicotiana tobacum ) cv. NC89 were used.
Site-directed mutagenesis of
SA-responsive cis-acting element
Polymerase chain reaction (PCR)-based site-directed mutagenesis of the two SA-responsive cis-acting elements (TGACG) in the VCH3 promoter was performed using the TaKaRa MutanBest kit (TaKaRa, Dalian, China). For the downstream SA cis-acting element present at -293 bp relative to the transcriptional start site, PCR was performed using the wild-type VCH3 promoter-inserted plasmid as the template, a mutant sense primer A, 5'-ATGCGGTTAACTCTTCCTAAG-3', and an antisense primer B, 5'-GCATTTTCTGACTCATTTCTC-3'. The single mutagenized nucleotide in the sense primer (underlined) resulted in the site mutation of the SA cis-acting element in the VCH3 promoter. After being blunted and 5' end phosphorylated, the resulting PCR products were then self-ligated and transformed into the E. coli cells, which resulted in the mutant VCH3 promoter containing one mutant SA cis-acting element. Similarly, the single point mutation was introduced in the upstream SA cis-acting element, which was located at -1181 bp relative to the transcriptional start site. PCR was performed using the mutant VCH3 promoter-insert plasmid as the template, a mutant sense primer C, 5'-TTGCGGTGTACTTTGGTTTTTG-3', and an antisense primer D, 5'-TGTGCTTGATTAATGTGTGTGAG-3'. Both mutations of the SA cis-acting elements in the VCH3 promoter were verified by sequencing.
Generation of the VCH3 promoter
deletion fragments and plasmid construction
To generate VCH3 promoter-GUS chimera, four wild-type and mutant VCH3 promoter deletion fragments were amplified by PCR using the wild-type or mutant VCH3 promoter-inserted plasmid as the template. A total of eight VCH3 promoter fragments from -1187, -892, -589, -276 to +7 bp relative to the transcriptional start site were generated using a common 3' oligonucleotide and different 5' oligonucleotides. The various deletion fragments of the VCH3 promoter were then cloned upstream of the GUS coding sequence in the binary vector pBI121. Briefly, PCR products of the VCH3 promoter fragments were blunted using Klenow fragment and were subsequently digested with BamHI. Plasmid pBI121 was cleaved with PstI, blunted using Klenow fragment, and then digested with BamHI. The blunt/BamHI VCH3 promoter fragments were inserted in the blunt/BamHI pBI121.
Transformation of tobacco
The GUS expression cassettes in the binary vector pBI121 were mobilized into A. tumefaciens strain LBA4404, which was kindly provided by the College of Life Sciences, Shandong Agricultural University. Leaf discs from N. tobacum cv. NC89 were transformed and plants were regenerated by standard methods [19]. For each construct, the presence of the GUS gene in transformed plants was verified by Southern blot analysis. Twelve independent transformant lines containing one copy of the chimeric gene were allowed to self-fertilize and seeds were collected and germinated on MS agar medium with 300 mg/ml kanamycin sulfate. Kanamycin-resistant T2 seedlings were used in the following induction.
SA-induction treatments
T2 seedlings at the 4-5 leaf stage were used in SA induction treatments. The tobacco plant roots were submerged in the MS medium without sucrose but supplemented with 1 mM SA at room temperature for 24 h before GUS activity was analyzed. In control plants, SA was replaced by distilled water.
Fluorometric assay of GUS
activity
Root samples were homogenized in 0.6 ml chilled lysis buffer containing 100 mM sodium phosphate (pH 7.0) and 1 M EDTA to obtain the crude homogenates, and 10 ml aliquots of the homogenates were used for measuring GUS activity by fluorometric assay as described by Jefferson et al. [20]. The activity was expressed as the specific activity in the crude homogenates, that is, pmol 4-methyl umbelliferone∙min-1∙(mg soluble protein)-1. Protein concentration was measured by the Bradford method [21] using bovine serum albumin as standard.
Histochemical analysis of GUS
activity
Histochemical analysis of GUS activity was performed as described by Stomp [22]. Tobacco roots 1-2 cm long were fixed by immersing them for 30 min in a fixing solution containing 100 mM sodium phosphate (pH 7.0), 0.1% formaldehyde, 0.1% Triton X-100 and 0.1% 2-mercaptoethanol. Fixed samples were stained by immersing the roots in a GUS staining solution containing 100 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.5 mM K ferrocyanide, 1 mM X-glucuronide and 0.1% Triton X-100. Tissues were vacuum infiltrated in the staining solution in order to assure homogeneous infection of the substrate. After dehydration in 100% ethanol, the tissues were incubated in toluene for 2 h at room temperature then embedded in paraffin for sectioning (8-10 mm in thickness). Pictures were taken from thin sections of tobacco roots under a microscope (BX 50; Olympus, Tokyo, Japan) equipped with the PM-30 automatic photomicrographic system (Olympus, Tokyo, Japan).
Results and Discussion
Promoter elements in the VCH3
promoter and generation of site-directed mutagenesis of the SA cis-acting
elements
The 1216 bp promoter sequence of chitinase gene VCH3 (GenBank accession number AF441123) was isolated and the transcriptional start site was identified by primer extension analysis [23]. Sequence analysis revealed that the VCH3 promoter contains two inverse SA cis-acting elements (TGACG) located at -293 and -1181 bp relative to the transcriptional start site (Fig. 1). In addition, four W-boxes [(T)TGAC(C/T)] [17-19] were found at -76, -591, -595 and -767 bp upstream of the transcriptional start site (Fig. 1).
To generate the mutant VCH3 promoter deletion fragments, PCR-based site-directed mutagenesis of the two SA cis-acting elements in the VCH3 promoter was performed (Fig. 2). The single mutagenized nucleotide present in the two sense primers resulted in the site mutation of the two inverse SA cis-acting elements (see "Materials and Methods"). Both mutations of the inverse SA cis-acting elements, from GCAGT to GCgGT in the VCH3 promoter, were verified by sequencing.
Construction of the VCH3
promoter fragment-GUS chimera and tobacco transformation
Four wild-type VCH3 promoter fragments and the corresponding four mutants with one or two mutated SA cis-acting element(s) were constructed upstream of the GUS coding region. This resulted in a total of eight constructs (Fig. 3), of which VCH3 (-1187)-GUS represented the maximal wild-type fragment containing two SA cis-acting elements and four W-boxes. m1VCH3 (-1187)-GUS and m2VCH3 (-1187)-GUS were the corresponding mutants which contained one and two mutated SA cis-acting element(s), respectively. Both the VCH3 (-892)-GUS and VCH3 (-589)-GUS fragments contained one SA cis-acting element, and four and one W-box(es), respectively, and mVCH3 (-892)-GUS and mVCH3 (-589)-GUS were the corresponding mutants, within which the SA cis-acting element was replaced by the mutated one. VCH3 (-276)-GUS represented the minimal fragment that contained only one W-box. All the constructs were transferred to N. tobacum cv. NC89 by A. tumefaciens-mediated leaf discs transformation, and 12 independent transgenic lines were obtained for each construct.
Fluorometric analysis of GUS
activities in transgenic tobacco roots induced by SA
GUS activity directed by the different VCH3 promoter fragments was investigated in the SA-treated roots of transgenic tobaccos transformed with the VCH3 promoter fragment-GUS constructs. Enhanced GUS activity was detected in the roots of the transgenic tobaccos transformed with the VCH3 (-1187)-GUS construct containing two SA-responsive cis-acting elements and four W-boxes (Fig. 4). However, GUS activity, directed by the -892 bp promoter fragment containing one SA cis-acting element and four W-boxes, decreased by up to 35% compared with that in tobaccos transformed with the VCH3 (-1187)-GUS construct. In addition, the m1VCH3 (-1187)-GUS construct, with mutation on one of the SA cis-acting elements within the VCH3 promoter fragment, reduced GUS activity to a level close to those found in transgenic plants transformed with the VCH3 (-892)-GUS construct, indicating the SA cis-acting element plays an important role in SA induction of the VCH3 promoter. It was noted that GUS activity in tobaccos transformed with the VCH3 (-589)-GUS construct containing one SA cis-acting element and one W-box showed a strong reduction compared with that in tobaccos transformed with the VCH3 (-892)-GUS construct, suggesting that the W-boxes also conferred the SA inducibility of the VCH3 promoter. However, the m2VCH3 (-1187)-GUS and mVCH3 (-892)-GUS constructs, with mutations on all of the SA cis-acting element(s) in their promoter fragment, did not abolish but showed a similar expression of GUS activity, further demonstrating that the W-boxes in the VCH3 promoter are involved in the activity of SA inducibility. Taken together, these results demonstrated that both the SA-responsive cis-acting elements and the W-boxes confer the SA induction of the VCH3 promoter.
The TGA family of transcription factors is known to bind to SA-responsive elements in the PR-1 promoter [9]. In addition, numerous in vitro and in vivo experiments have demonstrated that WRKY proteins specifically bind to the W-box in the promoters of those early defense-response genes [12-14]. Our present study demonstrated that the two SA-responsive elements and four W-boxes located in the VCH3 promoter (Fig. 1) were related to the activity of SA induction. Therefore, we suggest that some TGA family members of transcription factors and WRKY proteins might bind to the SA-responsive elements or W-boxes in the VCH3 promoter. Further analysis will be needed to decide how these cis elements interact with the TGA family of transcription factors or WRKY proteins to mediate the SA inducibility of the VCH3 promoter.
Histochemical analysis of GUS
activity in transgenic tobacco roots induced by SA
The transgenic tobacco roots expressing the VCH3 promoter fragment-GUS construct were treated with SA, and cross-sections were made from the resultant SA-treated tobacco transgenic roots (Fig. 5). GUS activities directed by all of the VCH3 promoter fragments were observed to be more active in vascular tissue than that in outer and inner cortexes, including the mutant VCH3 promoter deletion fragments containing one or two mutant SA cis-acting element(s). To date, many plant gene promoters induced by SA have been characterized [7-11]. However, few promoters have been reported to confer both SA-inducible and tissue-specific expression patterns [8]. In this study, we showed that the VCH3 promoter was strongly induced by SA and specifically expressed in tobacco vascular tissue, which indicated that efficient genetic engineering could be performed to design an appropriate gene expression system by using this SA induction of the VCH3 promoter to drive gene expression in vascular tissues when required. Also, and especially, this could drive the expression of plant disease-resistant genes, because many pathogens can spread rapidly throughout the plant once they penetrate the vascular system. Hence, this expression pattern of the VCH3 promoter might enhance the protection of such vulnerable tissues in response to pathogen infection.
Acknowledgements
We acknowledge Mr. Cheng-Chao ZHENG (Shandong Agricultural University, China) and Mr. Mark Andrew Hanson (Northeast Normal University, China) for their critical comments on the manuscript, and we would also like to express our appreciation to Mrs. Xiao-Lan NIE (Jilin Agricultural University, China) for her assistance with the histochemical analysis of GUS activity.
References
1�� Alvarez ME. Salicylic acid in the machinery of
hypersensitive cell death and disease resistance. Plant Mol Biol 2000, 44: 429-442
2�� Desveaux D, Subramaniam R, Despres C, Mess
JN, Levesque C, Fobert PR, Dangl JL et al. A �Whirly�
transcription factor is required for salicylic acid-dependent disease resistance
in Arabidopsis. Dev Cell 2004, 6: 229-240
3�� Delaney TP, Uknes S, Vernooij B, Friedrich L,
Weymann K, Negrotto D, Gaffney T et al. A central role of salicylic acid
in plant disease resistance. Science 1994, 266: 1247-1250
4�� Yalpani N, Enyedi AJ, Le�n J, Raskin I.
Ultraviolet light and ozone stimulate accumulation of salicylic acid,
pathogenesis-related proteins and virus resistance in tobacco. Planta 1994,
193: 372-376
5�� Stressmann M, Kitao S, Griffith M, Moresoli
C, Bravo LA, Marangoni AG. Calcium interacts with antifreeze proteins and
chitinase from cold-acclimated winter rye. Plant Physiol 2004, 135: 364-376
6�� Taji T, Seki M, Satou M, Sakurai T, Kobayashi
M, Ishiyama K, Narusaka Y et al. Comparative genomics in salt tolerance
between Arabidopsis and Arabidopsis-related halophyte salt cress
using Arabidopsis microarray. Plant Physiol 2004, 135: 1697-1709
7�� Chen W, Singh KB. The auxin, hydrogen
peroxide and salicylic acid induced expression of the Arabidopsis GST6
promoter is mediated in part by an ocs element. Plant J 1999, 19: 667-677
8�� Sa Q, Wang Y, Li W, Zhang L, Sun Y. The
promoter of an antifungal protein gene from Gastrodia elata confers
tissue-specific and fungus-inducible expression patterns and responds to both
salicylic acid and jasmonic acid. Plant Cell Rep 2003, 22: 79-84
9�� Gruner R, Strompen G, Pfitzner AJ, Pfitzner
UM. Salicylic acid and the hypersensitive response initiate distinct signal
transduction pathways in tobacco that converge on the as-1-like element
of the PR-1a promoter. Eur J Biochem 2003, 270: 4876-4886
10� Subramanian S, Hu X, Lu G, Odelland JT, Yu O.
The promoters of two
isoflavone synthase genes respond differentially to nodulation and defense
signals in transgenic soybean roots. Plant Mol Biol 2004, 54: 623-639
11� Yin Z, Hennig J, Szwacka M, Malepszy S.
Tobacco PR-2d promoter is induced in transgenic cucumber in response to
biotic and abiotic stimuli. J Plant Physiol 2004, 161: 621-629
12� Eulgem T, Rushton PJ, Schmelzer E, Hahlbrock K,
Somssich IE. Early nuclear events in plant defence signalling: Rapid gene
activation by WRKY transcription factors. EMBO J 1999, 18: 4689-4699
13� Du L, Chen Z. Identification of genes encoding
receptor-like protein kinases as possible targets of pathogen- and salicylic
acid-induced WRKY DNA-binding proteins in Arabidopsis. Plant J 2000, 24:
837-847
14� Yu D, Chen C, Chen Z. Evidence for an
important role of WRKY DNA binding proteins in the regulation of NPR1
gene expression. Plant Cell 2001, 13: 1527-1540
15� Chen C, Chen Z. Potentiation of
developmentally regulated plant defense response by AtWRKY18, a
pathogen-induced Arabidopsis transcription factor. Plant Physiol 2002,
129: 706-716
16� Dong J, Chen C, Chen Z. Expression profiles of
the Arabidopsis WRKY gene superfamily during plant defense response. Plant
Mol Biol 2003, 51: 21-37
17� Li J, Brader G, Palva ET. The WRKY70
transcription factor: A node of convergence for jasmonate-mediated and
salicylate-mediated signals in plant defense. Plant Cell 2004, 16: 319-331
18� Rocher A, Dumas C, Cock JM. A W-box is
required for full expression of the SA-responsive gene SFR2. Gene 2005, 344:
181-92
19� Horsch RB, Fry JE, Hoffmann NL, Eichholtz D,
Rogers SG, Fraley RT. A simple and general method for transferring genes into
plants. Science 1985, 227: 1229-1231
20� Jefferson RA, Kavanagh TA, Bevan MW. GUS
fusions: b-glucuronidase as a sensitive and versatile gene
fusion marker in higher plants. EMBO J 1987, 6: 3901-3907
21� Bradford MM. Rapid and sensitive methods for
quantitation of microgram quantities of protein utilizing the principle of
protein-dye binding. Annal Biochem 1976, 72: 248-254
22� Stomp AM. Histochemical location of b-glucuronidase. In:
Gallagher SR ed. GUS Protocols: Using the GUS Gene as a Reporter
of Gene Expression. San Diego: Academic Press 1992
23� Li HY, Qi J, Shu HR, Zheng CC, Li Y. Isolation and characterization of a chitinase gene VCH3 promoter from grapevine (Vitis amurensis). J Plant Physiol Mol Biol 2005, 31: 485-491