Biotechnol Lett DOI 10.1007/s10529-014-1553-y
ORIGINAL RESEARCH PAPER
Chimeric promoter mediates guard cell-specific gene expression in tobacco under water deficit Jong-Kuk Na • James D. Metzger
Received: 8 February 2014 / Accepted: 8 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The engineering of stomatal activity under water deficit through guard cell-specific gene regulation is an effective approach to improve drought tolerance of crops but it requires an appropriate promoter(s) inducible by water deficit in guard cells. We report that a chimeric promoter can induce guard cell-specific gene expression under water deficit. A chimeric promoter, p4xKST82-rd29B, was constructed using a tetramer of the 82 bp guard cellspecific regulatory region of potato KST1 promoter (4xKST82) and Arabidopsis dehydration-responsive rd29B promoter. Transgenic tobacco plants carrying p4xKST82-rd29B:mGFP-GUS exhibited GUS expression in response to water deficit. GUS enzyme activity of p4xKST82-rd29B:mGFP-GUS transgenic plants increased *300 % by polyethylene glycol treatment compared to that of control plant but not by abscisic acid (ABA), indicating that the p4xKST82-rd29B
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1553-y) contains supplementary material, which is available to authorized users. J.-K. Na (&) Division of Molecular Breeding, National Academy of Agricultural Science, RDA, Suwon 441-701, Republic of Korea e-mail: [email protected] J.-K. Na J. D. Metzger Department of Horticulture and Crop Science, Ohio State University, Columbus, OH 43210, USA e-mail: [email protected]
chimeric promoter can be used to induce the guard cell-specific expression of genes of interest in response to water deficit in an ABA-independent manner. Keywords Chimeric promoter Dehydration Guard cell KST1 rd29B Tobacco (transgenic) Water deficit
Introduction In higher plants, more than 90 % of water loss occurs through stomatal pores consisting of each pair of guard cells that regulate stomatal activity. Therefore, plant performance under water deficit can be improved by manipulating stomatal activity through guard cellspecific gene regulation. However, approaches to improve drought tolerance through guard cell-specific gene regulation have rarely been applied. This is likely due to the lack of an appropriate promoter that possesses both responsiveness to dehydration and guard cell specificity. Various guard cell-specific genes are associated with ion channels including K? influx or efflux channels. KST1 encodes K? influx channel protein and expresses specifically in guard cells (Plesch et al. 2001). A serial deletion assay of the KST1 promoter revealed that an 82 bp motif containing multiple TAAAAG sequences is crucial for guard cell-specific gene expression, and a tetramer of this motif was
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sufficient to induce strong reporter gene expression in guard cells (Plesch et al. 2001). In adaptive responses of plants to water deficit, the stress hormone, abscisic acid (ABA), is a pivotal molecule for reducing water loss by inducing stomatal closure and changing the expression of various stressresponsive genes including ABA-responsive genes. Well-studied prime examples among the ABAresponsive genes are rd29A and rd29B (Uno et al. 2000; Yamaguchi-Shinozaki and Shinozaki 1994). Detailed promoter analyses of rd29A and rd29B revealed that multiple ABA-responsive elements (ABREs) or the combination of ABRE and a coupling element (CE) are required for the expression of ABAresponsive genes (Narusaka et al. 2003; Yoshida et al. 2010). The rd29A promoter has been used successfully to induce genes of interest in response to water deficit (Kasuga et al. 1999, 2004) and salt stress (Jin et al. 2010). Given that rd29A and rd29B promoters can induce gene expression in response to ABA/dehydration and that the guard cell-specific region of KST1 promoter can induce guard cell-specific gene expression, combinatorial chimeric promoters consisting of these regulatory regions may be suitable for the induction of genes of interest in guard cells in response to water deficit. To test this, a series of chimeric promoters were constructed using 4xKST82, rd29A, rd29B promoter, and drought responsive element (DRE). Among chimeric promoters, p4xKST82-rd29B fusion promoter induced guard cell-specific reporter gene expression in response to water deficit, indicating that this promoter can be used as an invaluable tool to study or to induce genes of interest in guard cells under water deficit. Also, our results suggest that, in the absence of proper promoter to induce tissue-specific spatiotemporal gene regulation, chimeric promoter system is a very effective alternative way to achieve such gene regulation in plants.
containing MS medium supplemented with 30 g sucrose l-1, adjusted at pH 5.7, and solidified by 8 g select agar l-1 (Sigma). Seedlings were grown under 50 lmol m-2 s-1 of constant fluorescent light at 25 ± 3 °C. Promoter constructs Chimeric promoters were constructed using two or more of the following promoters: 281 bp rd29A promoter, 162 bp DRE of rd29A promoter, 345 bp rd29B promoter (Yamaguchi-Shinozaki and Shinozaki 1994), 298 bp KST1 promoter, a tetramer of 82 bp guard cell-specific region (designated to 4xKST82) of KST1 promoter (Plesch et al. 2001), 53 bp minimal CaMV 35S promoter (m35S), and 137 bp minimal KST1 promoters (mKST1). Each promoter or regulatory region was PCR-amplified using gene-specific primers (Supplementary Table S1) and used for the construction of a series of chimeric promoters. As briefly, PCR fragments of each promoter were cloned into BamHI/SalI or XbaI/ NcoI sites of 35S-sGFP expression vector (Chiu et al. 1996). Subsequently, chimeric promoters, p4xKST82rd29A, p4xKST82-rd29B, pDRE-4xKST82-m35S, and p4xKST82-m35S, were subcloned into HindIII/NcoI sites of pCambia-1304 binary vector. All inserts were confirmed by sequencing of plasmid DNAs using an Applied Biosystems 3730 DNA Analyzer. Transient expression assay and confocal laser microscopy Plasmid DNAs harboring the pKST1:sGFP, p4xKST82-mKST1:sGFP, or p35S:sGFP were transferred into 1-month-old tobacco leaves using the gene gun (Biorad PDS-1000/HE system). Two days after biolistic transformation, sGFP expression was observed under a PCM2000 confocal laser scanning microscope (Nikon).
Materials and methods
Plant transformation
Plant materials
For transformation, leaf explants of 1 month-old tobacco plants were pre-cultured on MS medium (2 % w/v sucrose, 8 g select agar l-1, pH 5.7) for 2 days. The pCambia 1304 binary vector harboring p4xKST82rd29A, p4xKST82-rd29B, p4xKST82-m35S, or pDRE4xKST82-m35S promoter was transformed into
Tobacco seeds (Nicotiana tabacum cv. Wiscosin 38) were surface-sterilized in 50 % (w/v) sodium hypochlorite solution for 10 min, followed by 4–5 rinses with sterile water, and placed in Magenta boxes
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Biotechnol Lett Fig. 1 Transient expression assay of pKST1:sGFP and p4xKST82-mKST1:sGFP constructs in tobacco leaves. a Schematic drawing of the expression vectors harboring p35S:sGFP, pKST1:sGFP, and p4xKST82-mKST1:sGFP cassettes. b Transient expression of the expression vectors in 1-month-old tobacco leaves. The expression vectors were transferred into tobacco leaves by Biolistic PDS1000/He particle delivery system (Bio-Rad). Tobacco leaves transfected with pKST1:sGFP were placed in the dark or under continuous light with/without 50 lM ABA for 2 days. sGFP expression was observed under a PCM2000 confocal laser scanning microscope (Nikon)
A
sGFP (S65T)
p35S
sGFP (S65T)
pKST1
sGFP (S65T)
p4xKST82-mKST1
B p35S
pKST1 (Light)
Agrobacterium tumefaciens strains GV3101 by electroporation. Overnight culture of A. tumefaciencs with each promoter:mGFP-GUS construct was used for tobacco leaf transformation as described by (Horsch 1989). Putative transformants were rooted on MS media containing 50 mg hygromycin l-1. The hygromycinresistant plantlets were transferred to 2 l pots with soilless media (Metro-Mix 360, The Scotts-Sierra Co., Marysville, OH, USA) and grown in a greenhouse with a 16 h/8 h day/night photoperiod. GUS enzyme activity and histochemical assay Three independent transgenic lines from each construct were used to examine GUS expression. Briefly, 2-week-old tobacco seedlings in MS medium (2 % w/v sucrose, 8 g select agar l-1, pH5.7) were transferred and grown in 200-cell square plug flat (16 cm3 for each cell) with Metro-Mix 360 in the greenhouse for 4 weeks. Histochemical GUS assay was carried out twice using leaves from each transgenic line by
p35S
pKST1 (ABA)
pKST1 (Dark)
p4xKST82-mKST1
following the GUS staining protocol (Jefferson et al. 1987). As for GUS enzyme activity, 2-week-old seedlings of p4xKST82-rd29B:mGFP-GUS transgenic plants grown in solid MS media were transferred to liquid MS media containing 50 lM ABA, 200 mM NaCl, or 20 % polyethylene glycol (PEG) to examine GUS activity under various stress conditions. Proteins were extracted from the seedlings treated with various stresses for 24 h and used for GUS enzyme activity by fluorometric quantitation of 4-methylumbelliferone produced from the glucuronide precursor using a standard protocol (Jefferson et al. 1986).
Results and discussion Because transpiration accounts for most of water loss in plants, the modulation of stomatal activity through the guard cell-specific gene regulation can be a useful tool to improve drought tolerance of crops (Schroeder et al. 2001). However, such approaches have rarely
123
Biotechnol Lett Fig. 2 Construction of the guard cell-specific and dehydration-responsive chimeric promoters. a TDNA region of pCambia 1304 binary vectors harboring chimeric promoter constructs consisting of two or more of the following promoters or regulatory regions: 358 bp guard cell-specific 4xKST82, 281 bp rd29A promoter, 162 bp dehydration-responsive element (DRE), 345 bp rd29B promoter, 298 bp KST1 promoter, 53 bp minimal 35S promoter (m35S). The pCambia 1304 binary vectors carrying chimeric promoters, p4xKST82-rd29A, p4xKST82-m35S, pDRE4xKST82-m35, and p4xKST82-rd29B, were transformed into tobacco plant. b GUS expression was examined in guard cells of transgenic tobacco plants grown in greenhouse. Three independent transgenic lines from each construct were used for GUS staining
A 1
p4xKST82-rd29A
2
p4xKST82-m35S
mGFP-GUS
3
pDRE-4xKST82-m35S
mGFP-GUS
4
p4xKST82-rd29B
mGFP-GUS
B 1
3
2
4
been attempted likely due to the lack of a promoter that can induce temporal gene expression in guard cells under water deficit. Therefore, alternative approach should be taken to achieve guard cell-mediated stomatal regulation under water deficit. Accordingly, this study was aimed to generate chimeric promoters, consisting of previously reported promoters or their regulatory regions, which can induce target gene expression in guard cells under water deficit.
Construction of a guard cell-specific and dehydration-responsive chimeric promoter Prior to constructing chimeric promoters, the guard cell specificity of pKST1 and p4xKST82-mKST1 promoters were examined by transiently expressing pKST1:sGFP and p4xKST82-mKST1:sGFP constructs in tobacco leaves (Fig. 1a). Both promoters induced strong guard cell-specific GFP expression (Fig. 1b),
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mGFP-GUS
which is consistent with previous report (Plesch et al. 2001). Given that KST1 gene encodes K? influx channel protein and that K? influx requires stomatal opening, KST1 expression may increase in response to light but decrease by darkness and ABA. To test this, tobacco leaves transfected by pKST1:sGFP construct were placed under continuous light or darkness, or on the top of water containing 50 lM ABA under continuous light for 2 days. However, GFP expression was comparable among treatments (Fig. 1b), suggesting that KST1 promoter is not sensitive to such changes. To construct chimeric promoters, the guard cellspecificity was derived from 4xKST82 (Plesch et al. 2001) and the responsiveness to water deficit from Arabidopsis rd29A or rd29B promoter, or DRE element (Yamaguchi-Shinozaki and Shinozaki 1994). Chimeric promoters were generated by combinatorial orders of rd29A, DRE, rd29B, 4xKST82, or 53 bp minimal 35S promoters (m35S). Four chimeric
Biotechnol Lett Fig. 3 The p4xKST82rd29B fusion promoter induces guard cell-specific gene expression under water deficit. a GUS expression under non-stressed or dehydration conditions. b GUS expression in three independent p4xKST82rd29B:mGFP-GUS transgenic lines under nonstressed or water deficit conditions. GUS transcripts were analyzed by RT-PCR using gene-specific primers. c GUS enzyme activity of the 4xKST82-rd29B fusion promoter under various stress conditions: 50 lM ABA, 20 % PEG, or 200 mM NaCl. GUS enzyme activity assay was repeated three times and error bars denote standard deviation
p4xKST82-rd29B:mGFP-GUS Dehydration Non-stressed
A
Non-stressed 1 2 3
B
Dehydration 1 2 3
GUS rRNA
C
3000
GUS enzyme activity
4MU pmol/ug protein/min
2500
2000
1500
1000
500
0 MS
fusion promoters, p4xKST82-rd29A (#1), p4xKST82m35S (#2), pDRE-4xKST82-m35 (#3), and p4xKST82rd29B (#4), were generated and cloned into the upstream of mGFP-GUS reporter gene in pCambia 1304 binary vector (Fig. 2a). Resulting constructs were transformed into tobacco plants. Three independent transgenic lines from each construct were grown in well-watered conditions in greenhouse for 1 month
50 uM ABA
20 % PEG
200 mM NaCl
and leaves from each transgenic line were harvested for GUS staining. Strong GUS accumulation was detected in guard cells of transgenic lines containing construct #1, #2, or #3, while very weak GUS expression was detected from transgenic lines with construct #4 (Fig. 2b). These results strongly indicate that three chimeric promoters, #1, #2, and #3, can induce target gene
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expression in guard cells under non-stressed conditions. Therefore, construct #4, p4xKST82-rd29B, was selected and used for further study. The p4xKST82-rd29B promoter responds to dehydration in guard cells To investigate the activity of the p4xKST82-rd29B promoter, p4xKST82-rd29B:mGFP-GUS transgenic plants were subjected to water deficit and examined for GUS expression. GUS accumulation was detected in guard cells of the p4xKST82-rd29B:mGFP-GUS transgenic plants under water deficit, whereas it was barely detectable from non-stressed transgenic plants (Fig. 3a). Total RNAs were extracted from waterstressed and non-stressed tobacco leaves of p4xKST82-rd29B:mGFP-GUS transgenic plants and used for RT-PCR analysis to examine GUS expression using gene-specific primers (Supplementary Table 1). GUS expression in p4xKST82-rd29B:mGFP-GUS plants was upregulated by water deficit (Fig. 3b), which coincides with GUS accumulation as shown in Fig. 3a. In addition, GUS enzyme activities of the p4xKST82-rd29B:mGFP-GUS transgenic plants were examined in various stress conditions: 50 lM ABA, 20 % PEG, or 200 mM NaCl. GUS activity of p4xKST82-rd29B:mGFP-GUS transgenic plants increased *300 % under water deficit (PEG) but not by 50 lM ABA and 200 mM NaCl (Fig. 3c), indicating that p4xKST82-rd29B promoter is responsive to water deficit in ABA-independent manner. Given that rd29B promoter contains several ABRE elements (Msanne et al. 2011) and that the expression of rd29B is ABA-dependent in Arabidopsis (Yamaguchi-Shinozaki and Shinozaki 1994; Msanne et al. 2011), it is intriguing that p4xKST82rd29B promoter did not respond to exogenous ABA (Fig. 3c). One of possible explanations could be because ABRE elements in Arabidopsis rd29B promoter are less or not sensitive to ABA in tobacco. Another possibility can be that the induction of rd29B may differ at various developmental stages. In Arabidopsis, exogenous ABA application can induce strong rd29B expression in 1-week-old seedlings but not in 2-week-old plant (Nakashima et al. 2006). Given that the expression of rd29B differs along with developmental stages, it can be speculated that 1 month-old transgenic tobacco plants that we used for GUS
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enzyme activity may be not responsive to ABA at that stage. Rusconi et al. (2013) reported that they had developed an ABA-/dehydration-inducible and guard cell-specific synthetic promoter using the minimal AtMYB60 promoter (MYB60pro246) as a source for guard cell specific module and rd29A as a source for ABA-/dehydration-inducible module. In the study, rd29A promoter was fused to the upstream of guard cell-specific MYB60pro246 promoter (Cominelli et al. 2011) and the resulting rd29A-MYB60pro246 promoter then directed ABA-/dehydration-inducible gene expression in guard cells (Rusconi et al. 2013). Both the rd29A-MYB60pro246 and our p4xKST82-rd29B promoter directed guard cell-specific gene expression regardless of the order of guard cell-specific module and stress-inducible module. These results suggest that a chimeric promoter can be generated relatively easily and is an effective alternative to achieve spatiotemporal gene regulation in the absence of proper promoter. Acknowledgments We are most grateful to Dr. JC Jang for providing his lab area and various materials. We also thank Dr. Biao Ding for access to his confocal microscope. This research was partially supported by the OARDC Research Enhancement Competitive Grant Program to JKN.
References Chiu WL, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330 Cominelli E, Galbiati M, Albertini A, Fornara F, Conti L, Coupland G, Tonelli C (2011) DOF-binding sites additively contribute to guard cell-specificity of AtMYB60 promoter. BMC Plant Biol 11:162 Horsch RB, Fry J, Hoffmann N, Neidermeyer J, Rogers SG, Fraley RT (1989) Leaf disc transformation. Plant molecular biology manual. Springer, Dordrecht Jefferson RA, Burgess SM, Hirsh D (1986) Beta-glucuronidase from Escherichia coli as a gene-fusion marker. Proc Nat Acad Sci USA 83:8447–8451 Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907 Jin TC, Chang Q, Li WF, Yin DX, Li ZJ, Wang DL, Liu B, Liu LX (2010) Stress-inducible expression of GmDREB1 conferred salt tolerance in transgenic alfalfa. Plant Cell Tissue Organ 100:219–227 Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Improving plant drought, salt, and freezing
Biotechnol Lett tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol 17:287–291 Kasuga M, Miura S, Shinozaki K, Yamaguchi-Shinozaki K (2004) A combination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved droughtand low-temperature stress tolerance in tobacco by gene transfer. Plant Cell Physiol 45:346–350 Msanne J, Lin J, Stone JM, Awada T (2011) Characterization of abiotic stress-responsive Arabidopsis thaliana RD29A and RD29B genes and evaluation of transgenes. Planta 234: 97–107 Nakashima K, Fujita Y, Katsura K, Maruyama K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2006) Transcriptional regulation of ABI3- and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of Arabidopsis. Plant Mol Biol 60:51–68 Narusaka Y, Nakashima K, Shinwari ZK, Sakuma Y, Furihata T, Abe H, Narusaka M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and highsalinity stresses. Plant J 34:137–148 Plesch G, Ehrhardt T, Mueller-Roeber B (2001) Involvement of TAAAG elements suggests a role for Dof transcription factors in guard cell-specific gene expression. Plant J 28:455–464
Rusconi F, Simeoni F, Francia P, Cominelli E, Conti L, Riboni M, Simoni L, Martin CR, Tonelli C, Galbiati M (2013) The Arabidopsis thaliana MYB60 promoter provides a tool for the spatio-temporal control of gene expression in stomatal guard cells. J Exp Bot 64:3361–3371 Schroeder JI, Kwak JM, Allen GJ (2001) Guard cell abscisic acid signalling and engineering drought hardiness in plants. Nature 410:327–330 Uno Y, Furihata T, Abe H, Yoshida R, Shinozaki K, Yamaguchi-Shinozaki K (2000) Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and highsalinity conditions. Proc Natl Acad Sci USA 97: 11632–11637 Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6:251–264 Yoshida T, Fujita Y, Sayama H, Kidokoro S, Maruyama K, Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2010) AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent ABA signaling involved in drought stress tolerance and require ABA for full activation. Plant J 61:672–685
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ORIGINAL RESEARCH PAPER
Chimeric promoter mediates guard cell-specific gene expression in tobacco under water deficit Jong-Kuk Na • James D. Metzger
Received: 8 February 2014 / Accepted: 8 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The engineering of stomatal activity under water deficit through guard cell-specific gene regulation is an effective approach to improve drought tolerance of crops but it requires an appropriate promoter(s) inducible by water deficit in guard cells. We report that a chimeric promoter can induce guard cell-specific gene expression under water deficit. A chimeric promoter, p4xKST82-rd29B, was constructed using a tetramer of the 82 bp guard cellspecific regulatory region of potato KST1 promoter (4xKST82) and Arabidopsis dehydration-responsive rd29B promoter. Transgenic tobacco plants carrying p4xKST82-rd29B:mGFP-GUS exhibited GUS expression in response to water deficit. GUS enzyme activity of p4xKST82-rd29B:mGFP-GUS transgenic plants increased *300 % by polyethylene glycol treatment compared to that of control plant but not by abscisic acid (ABA), indicating that the p4xKST82-rd29B
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1553-y) contains supplementary material, which is available to authorized users. J.-K. Na (&) Division of Molecular Breeding, National Academy of Agricultural Science, RDA, Suwon 441-701, Republic of Korea e-mail: [email protected] J.-K. Na J. D. Metzger Department of Horticulture and Crop Science, Ohio State University, Columbus, OH 43210, USA e-mail: [email protected]
chimeric promoter can be used to induce the guard cell-specific expression of genes of interest in response to water deficit in an ABA-independent manner. Keywords Chimeric promoter Dehydration Guard cell KST1 rd29B Tobacco (transgenic) Water deficit
Introduction In higher plants, more than 90 % of water loss occurs through stomatal pores consisting of each pair of guard cells that regulate stomatal activity. Therefore, plant performance under water deficit can be improved by manipulating stomatal activity through guard cellspecific gene regulation. However, approaches to improve drought tolerance through guard cell-specific gene regulation have rarely been applied. This is likely due to the lack of an appropriate promoter that possesses both responsiveness to dehydration and guard cell specificity. Various guard cell-specific genes are associated with ion channels including K? influx or efflux channels. KST1 encodes K? influx channel protein and expresses specifically in guard cells (Plesch et al. 2001). A serial deletion assay of the KST1 promoter revealed that an 82 bp motif containing multiple TAAAAG sequences is crucial for guard cell-specific gene expression, and a tetramer of this motif was
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Biotechnol Lett
sufficient to induce strong reporter gene expression in guard cells (Plesch et al. 2001). In adaptive responses of plants to water deficit, the stress hormone, abscisic acid (ABA), is a pivotal molecule for reducing water loss by inducing stomatal closure and changing the expression of various stressresponsive genes including ABA-responsive genes. Well-studied prime examples among the ABAresponsive genes are rd29A and rd29B (Uno et al. 2000; Yamaguchi-Shinozaki and Shinozaki 1994). Detailed promoter analyses of rd29A and rd29B revealed that multiple ABA-responsive elements (ABREs) or the combination of ABRE and a coupling element (CE) are required for the expression of ABAresponsive genes (Narusaka et al. 2003; Yoshida et al. 2010). The rd29A promoter has been used successfully to induce genes of interest in response to water deficit (Kasuga et al. 1999, 2004) and salt stress (Jin et al. 2010). Given that rd29A and rd29B promoters can induce gene expression in response to ABA/dehydration and that the guard cell-specific region of KST1 promoter can induce guard cell-specific gene expression, combinatorial chimeric promoters consisting of these regulatory regions may be suitable for the induction of genes of interest in guard cells in response to water deficit. To test this, a series of chimeric promoters were constructed using 4xKST82, rd29A, rd29B promoter, and drought responsive element (DRE). Among chimeric promoters, p4xKST82-rd29B fusion promoter induced guard cell-specific reporter gene expression in response to water deficit, indicating that this promoter can be used as an invaluable tool to study or to induce genes of interest in guard cells under water deficit. Also, our results suggest that, in the absence of proper promoter to induce tissue-specific spatiotemporal gene regulation, chimeric promoter system is a very effective alternative way to achieve such gene regulation in plants.
containing MS medium supplemented with 30 g sucrose l-1, adjusted at pH 5.7, and solidified by 8 g select agar l-1 (Sigma). Seedlings were grown under 50 lmol m-2 s-1 of constant fluorescent light at 25 ± 3 °C. Promoter constructs Chimeric promoters were constructed using two or more of the following promoters: 281 bp rd29A promoter, 162 bp DRE of rd29A promoter, 345 bp rd29B promoter (Yamaguchi-Shinozaki and Shinozaki 1994), 298 bp KST1 promoter, a tetramer of 82 bp guard cell-specific region (designated to 4xKST82) of KST1 promoter (Plesch et al. 2001), 53 bp minimal CaMV 35S promoter (m35S), and 137 bp minimal KST1 promoters (mKST1). Each promoter or regulatory region was PCR-amplified using gene-specific primers (Supplementary Table S1) and used for the construction of a series of chimeric promoters. As briefly, PCR fragments of each promoter were cloned into BamHI/SalI or XbaI/ NcoI sites of 35S-sGFP expression vector (Chiu et al. 1996). Subsequently, chimeric promoters, p4xKST82rd29A, p4xKST82-rd29B, pDRE-4xKST82-m35S, and p4xKST82-m35S, were subcloned into HindIII/NcoI sites of pCambia-1304 binary vector. All inserts were confirmed by sequencing of plasmid DNAs using an Applied Biosystems 3730 DNA Analyzer. Transient expression assay and confocal laser microscopy Plasmid DNAs harboring the pKST1:sGFP, p4xKST82-mKST1:sGFP, or p35S:sGFP were transferred into 1-month-old tobacco leaves using the gene gun (Biorad PDS-1000/HE system). Two days after biolistic transformation, sGFP expression was observed under a PCM2000 confocal laser scanning microscope (Nikon).
Materials and methods
Plant transformation
Plant materials
For transformation, leaf explants of 1 month-old tobacco plants were pre-cultured on MS medium (2 % w/v sucrose, 8 g select agar l-1, pH 5.7) for 2 days. The pCambia 1304 binary vector harboring p4xKST82rd29A, p4xKST82-rd29B, p4xKST82-m35S, or pDRE4xKST82-m35S promoter was transformed into
Tobacco seeds (Nicotiana tabacum cv. Wiscosin 38) were surface-sterilized in 50 % (w/v) sodium hypochlorite solution for 10 min, followed by 4–5 rinses with sterile water, and placed in Magenta boxes
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Biotechnol Lett Fig. 1 Transient expression assay of pKST1:sGFP and p4xKST82-mKST1:sGFP constructs in tobacco leaves. a Schematic drawing of the expression vectors harboring p35S:sGFP, pKST1:sGFP, and p4xKST82-mKST1:sGFP cassettes. b Transient expression of the expression vectors in 1-month-old tobacco leaves. The expression vectors were transferred into tobacco leaves by Biolistic PDS1000/He particle delivery system (Bio-Rad). Tobacco leaves transfected with pKST1:sGFP were placed in the dark or under continuous light with/without 50 lM ABA for 2 days. sGFP expression was observed under a PCM2000 confocal laser scanning microscope (Nikon)
A
sGFP (S65T)
p35S
sGFP (S65T)
pKST1
sGFP (S65T)
p4xKST82-mKST1
B p35S
pKST1 (Light)
Agrobacterium tumefaciens strains GV3101 by electroporation. Overnight culture of A. tumefaciencs with each promoter:mGFP-GUS construct was used for tobacco leaf transformation as described by (Horsch 1989). Putative transformants were rooted on MS media containing 50 mg hygromycin l-1. The hygromycinresistant plantlets were transferred to 2 l pots with soilless media (Metro-Mix 360, The Scotts-Sierra Co., Marysville, OH, USA) and grown in a greenhouse with a 16 h/8 h day/night photoperiod. GUS enzyme activity and histochemical assay Three independent transgenic lines from each construct were used to examine GUS expression. Briefly, 2-week-old tobacco seedlings in MS medium (2 % w/v sucrose, 8 g select agar l-1, pH5.7) were transferred and grown in 200-cell square plug flat (16 cm3 for each cell) with Metro-Mix 360 in the greenhouse for 4 weeks. Histochemical GUS assay was carried out twice using leaves from each transgenic line by
p35S
pKST1 (ABA)
pKST1 (Dark)
p4xKST82-mKST1
following the GUS staining protocol (Jefferson et al. 1987). As for GUS enzyme activity, 2-week-old seedlings of p4xKST82-rd29B:mGFP-GUS transgenic plants grown in solid MS media were transferred to liquid MS media containing 50 lM ABA, 200 mM NaCl, or 20 % polyethylene glycol (PEG) to examine GUS activity under various stress conditions. Proteins were extracted from the seedlings treated with various stresses for 24 h and used for GUS enzyme activity by fluorometric quantitation of 4-methylumbelliferone produced from the glucuronide precursor using a standard protocol (Jefferson et al. 1986).
Results and discussion Because transpiration accounts for most of water loss in plants, the modulation of stomatal activity through the guard cell-specific gene regulation can be a useful tool to improve drought tolerance of crops (Schroeder et al. 2001). However, such approaches have rarely
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Biotechnol Lett Fig. 2 Construction of the guard cell-specific and dehydration-responsive chimeric promoters. a TDNA region of pCambia 1304 binary vectors harboring chimeric promoter constructs consisting of two or more of the following promoters or regulatory regions: 358 bp guard cell-specific 4xKST82, 281 bp rd29A promoter, 162 bp dehydration-responsive element (DRE), 345 bp rd29B promoter, 298 bp KST1 promoter, 53 bp minimal 35S promoter (m35S). The pCambia 1304 binary vectors carrying chimeric promoters, p4xKST82-rd29A, p4xKST82-m35S, pDRE4xKST82-m35, and p4xKST82-rd29B, were transformed into tobacco plant. b GUS expression was examined in guard cells of transgenic tobacco plants grown in greenhouse. Three independent transgenic lines from each construct were used for GUS staining
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been attempted likely due to the lack of a promoter that can induce temporal gene expression in guard cells under water deficit. Therefore, alternative approach should be taken to achieve guard cell-mediated stomatal regulation under water deficit. Accordingly, this study was aimed to generate chimeric promoters, consisting of previously reported promoters or their regulatory regions, which can induce target gene expression in guard cells under water deficit.
Construction of a guard cell-specific and dehydration-responsive chimeric promoter Prior to constructing chimeric promoters, the guard cell specificity of pKST1 and p4xKST82-mKST1 promoters were examined by transiently expressing pKST1:sGFP and p4xKST82-mKST1:sGFP constructs in tobacco leaves (Fig. 1a). Both promoters induced strong guard cell-specific GFP expression (Fig. 1b),
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which is consistent with previous report (Plesch et al. 2001). Given that KST1 gene encodes K? influx channel protein and that K? influx requires stomatal opening, KST1 expression may increase in response to light but decrease by darkness and ABA. To test this, tobacco leaves transfected by pKST1:sGFP construct were placed under continuous light or darkness, or on the top of water containing 50 lM ABA under continuous light for 2 days. However, GFP expression was comparable among treatments (Fig. 1b), suggesting that KST1 promoter is not sensitive to such changes. To construct chimeric promoters, the guard cellspecificity was derived from 4xKST82 (Plesch et al. 2001) and the responsiveness to water deficit from Arabidopsis rd29A or rd29B promoter, or DRE element (Yamaguchi-Shinozaki and Shinozaki 1994). Chimeric promoters were generated by combinatorial orders of rd29A, DRE, rd29B, 4xKST82, or 53 bp minimal 35S promoters (m35S). Four chimeric
Biotechnol Lett Fig. 3 The p4xKST82rd29B fusion promoter induces guard cell-specific gene expression under water deficit. a GUS expression under non-stressed or dehydration conditions. b GUS expression in three independent p4xKST82rd29B:mGFP-GUS transgenic lines under nonstressed or water deficit conditions. GUS transcripts were analyzed by RT-PCR using gene-specific primers. c GUS enzyme activity of the 4xKST82-rd29B fusion promoter under various stress conditions: 50 lM ABA, 20 % PEG, or 200 mM NaCl. GUS enzyme activity assay was repeated three times and error bars denote standard deviation
p4xKST82-rd29B:mGFP-GUS Dehydration Non-stressed
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fusion promoters, p4xKST82-rd29A (#1), p4xKST82m35S (#2), pDRE-4xKST82-m35 (#3), and p4xKST82rd29B (#4), were generated and cloned into the upstream of mGFP-GUS reporter gene in pCambia 1304 binary vector (Fig. 2a). Resulting constructs were transformed into tobacco plants. Three independent transgenic lines from each construct were grown in well-watered conditions in greenhouse for 1 month
50 uM ABA
20 % PEG
200 mM NaCl
and leaves from each transgenic line were harvested for GUS staining. Strong GUS accumulation was detected in guard cells of transgenic lines containing construct #1, #2, or #3, while very weak GUS expression was detected from transgenic lines with construct #4 (Fig. 2b). These results strongly indicate that three chimeric promoters, #1, #2, and #3, can induce target gene
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expression in guard cells under non-stressed conditions. Therefore, construct #4, p4xKST82-rd29B, was selected and used for further study. The p4xKST82-rd29B promoter responds to dehydration in guard cells To investigate the activity of the p4xKST82-rd29B promoter, p4xKST82-rd29B:mGFP-GUS transgenic plants were subjected to water deficit and examined for GUS expression. GUS accumulation was detected in guard cells of the p4xKST82-rd29B:mGFP-GUS transgenic plants under water deficit, whereas it was barely detectable from non-stressed transgenic plants (Fig. 3a). Total RNAs were extracted from waterstressed and non-stressed tobacco leaves of p4xKST82-rd29B:mGFP-GUS transgenic plants and used for RT-PCR analysis to examine GUS expression using gene-specific primers (Supplementary Table 1). GUS expression in p4xKST82-rd29B:mGFP-GUS plants was upregulated by water deficit (Fig. 3b), which coincides with GUS accumulation as shown in Fig. 3a. In addition, GUS enzyme activities of the p4xKST82-rd29B:mGFP-GUS transgenic plants were examined in various stress conditions: 50 lM ABA, 20 % PEG, or 200 mM NaCl. GUS activity of p4xKST82-rd29B:mGFP-GUS transgenic plants increased *300 % under water deficit (PEG) but not by 50 lM ABA and 200 mM NaCl (Fig. 3c), indicating that p4xKST82-rd29B promoter is responsive to water deficit in ABA-independent manner. Given that rd29B promoter contains several ABRE elements (Msanne et al. 2011) and that the expression of rd29B is ABA-dependent in Arabidopsis (Yamaguchi-Shinozaki and Shinozaki 1994; Msanne et al. 2011), it is intriguing that p4xKST82rd29B promoter did not respond to exogenous ABA (Fig. 3c). One of possible explanations could be because ABRE elements in Arabidopsis rd29B promoter are less or not sensitive to ABA in tobacco. Another possibility can be that the induction of rd29B may differ at various developmental stages. In Arabidopsis, exogenous ABA application can induce strong rd29B expression in 1-week-old seedlings but not in 2-week-old plant (Nakashima et al. 2006). Given that the expression of rd29B differs along with developmental stages, it can be speculated that 1 month-old transgenic tobacco plants that we used for GUS
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enzyme activity may be not responsive to ABA at that stage. Rusconi et al. (2013) reported that they had developed an ABA-/dehydration-inducible and guard cell-specific synthetic promoter using the minimal AtMYB60 promoter (MYB60pro246) as a source for guard cell specific module and rd29A as a source for ABA-/dehydration-inducible module. In the study, rd29A promoter was fused to the upstream of guard cell-specific MYB60pro246 promoter (Cominelli et al. 2011) and the resulting rd29A-MYB60pro246 promoter then directed ABA-/dehydration-inducible gene expression in guard cells (Rusconi et al. 2013). Both the rd29A-MYB60pro246 and our p4xKST82-rd29B promoter directed guard cell-specific gene expression regardless of the order of guard cell-specific module and stress-inducible module. These results suggest that a chimeric promoter can be generated relatively easily and is an effective alternative to achieve spatiotemporal gene regulation in the absence of proper promoter. Acknowledgments We are most grateful to Dr. JC Jang for providing his lab area and various materials. We also thank Dr. Biao Ding for access to his confocal microscope. This research was partially supported by the OARDC Research Enhancement Competitive Grant Program to JKN.
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