Journal of Integrative Agriculture 2015, 14(1): 1–10 Available online at www.sciencedirect.com
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RESEARCH ARTICLE
Identification and validation of root-specific promoters in rice HUANG Li-yu, ZHANG Fan, QIN Qiao, WANG Wen-sheng, ZHANG Ting, FU Bin-ying National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China
Abstract Novel promoters that confer root-specific expression would be useful for engineering resistance against problems of nutrient and water absorption by roots. In this study, the reverse transcriptase polymerase chain reaction was used to identify seven genes with root-specific expression in rice. The isolation and characterization of upstream promoter regions of five selected genes rice root-specific promoter (rRSP) 1 to 5 (rRSP1-rRSP5) and A2P (the promoter of OsAct2) revealed that rRSP1, rRSP3, and rRSP5 are particularly important with respect to root-specific activities. Furthermore, rRSP1, rRSP3, and rRSP5 were observed to make different contributions to root activities in various species. These three promoters could be used for root-specific enhancement of target gene(s). Keywords: rice, root-specific promoters, expression profile
1. Introduction In dicotyledonous and monocotyledonous plants, high-level expression may be required in certain tissues, such as roots, and may be achieved by constitutive promoters. In other cases, the expression of transgenes that confer desired traits might be minimized, especially if the expression of the transgenes is restricted to required tissues, and genes confer to detrimental, tissue-specific functional products. Ubiquitous expression is often associated with undesirable phenotypes, including growth retardation, delayed flowering and even lethality or sterility (Gilmour et al. 2000; Hsieh et al. 2002; Kasuga et al. 2004; Benedict et al. 2006; Pino
Received 3 January, 2014 Accepted 14 April, 2014 HUANG Li-yu, E-mail:
[email protected]; Correspondence FU Bin-ying, Tel: +86-10-82106698, Fax: +86-10-82108559, E-mail:
[email protected] © 2015, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(14)60763-2
et al. 2007). Tissue-specific promoters have the potential to avoid the harm from overexpression of target genes that are under the control of constitutive promoters in non-targeted organs of transgenic plants. To date, both exogenous constitutive promoters, e.g., 35S derived from cauliflower mosaic virus (CaMV), and a few native constitutive promoters that are well characterized in plants, e.g., the promoters of OsAct1 (McElroy et al. 1991), Actin2 gene (OsAct2) (He et al. 2009), PGD1 (Park et al. 2010), α-tubulin gene (OsTubA1) (Jeon et al. 2000), OsCc1 (Jang et al. 2002), polyubiquitin rubi1, 2, 3 genes (RUBQ1, 2 and rubi3) (Wang and Oard 2003; Lu et al. 2008), and polyubiquitin genes (ZmUbi1) (Christensen et al. 1992; Cornejo et al. 1993), have been widely used. Precise regulation of gene expression is essential for all organisms during tissue development and environmental adaptation. The temporal and spatial expression patterns of genes are controlled mainly by short cis-regulatory elements that contain binding sites for transcription factors (Zhao et al. 2007). The CaMV 35S promoter is highly constitutive, but Domain A (90 bp upstream) of the 35S promoter is an
HUANG Li-yu et al. Journal of Integrative Agriculture 2015, 14(1): 1–10
177-103 and DK151 are salt and drought tolerance introgression lines, respectively. IR64 is their recurrent parent. CK, untreated control. ABA, abscisic acid. 1)
Drought 1 206.12 841.30 75.93 487.75 2 254.51 165.85 518.71 Salt +ABA CK 1 026.14 2 036.17 5 641.37 1 529.90 487.75 141.00 416.39 485.89 5 035.87 2 452.02 339.36 355.86 1 268.86 282.64 Salt 723.61 7 774.54 327.53 555.91 4 159.78 260.28 1 772.57 Salt +ABA CK 776.55 835.62 5 452.14 8 054.54 455.31 380.75 360.91 403.22 4 854.83 4 810.91 255.01 190.56 1 164.13 950.58 Salt 654.47 7 430.41 364.99 481.41 4 169.63 196.14 1 507.98 CK 846.91 8 202.99 386.39 321.55 4 750.27 133.65 913.61 Os02g0658100 Os03g0107300 Os07g0646800 Os05g0148900 Os04g0423800 Os06g0500700 Os03g0185700
rRSP1 rRSP2 rRSP3 rRSP4 rRSP5 rRSP6 rRSP7
177-103/Seedling stage IR64/Seedling stage
Promoter name
Os.12345.1.S1_at Os.18734.1.S1_at Os.34356.1.S1_at Os.50371.1.S1_x_at Os.5130.1.S1_at OsAffx.27884.1.S1_at Os.55882.1.S1_at
We mined the transcriptome datasets to identify genes with high expression in roots and low expression in all other organs under a wide range of growth conditions. According to the microarray data, seven genes that are highly and specifically expressed in roots were selected for further analysis (Table 1). At the transcript level, the expressions of the seven genes were undetectable in other tissues (labeled as absent), but stably expressed in root tissues under drought and salt stresses (Table 1). The expressions of the seven root-specific genes (Os02g0658100, Os03g0107300,
RSG1 RSG2 RSG3 RSG4 RSG5 RSG6 RSG7
2.1. Identification of novel root-specific genes in rice
Gene ID
2. Results
Probe set ID
expression in their corresponding transgenic plants (Elmayan and Tepfer 1995). A few root-specific genes have been isolated from a wide variety of plants and their promoters have been used to drive root-specific gene expression in transgenic plants. For example, the tobacco TobRB7 and rice RCc3 promoters have been widely used for the genetic engineering of abiotic resistance (Yamamoto et al. 1991; Xu et al. 1995; Jeong et al. 2010). Recently, a study characterized the novel root-specific promoters Os03g01700 and Os02g37190, which are highly expressed in root tissues and provide useful alternatives for root-specific transformations in rice (Li et al. 2013). Although efforts have been made to characterize root-specific genes and their promoters, root-specific promoters containing different activities and that are highly conserved across species need to be further identified. Additionally, differences in the transformation efficiency of transgenes driven by the same promoter in different species also indicate the necessity to develop endogenous root-specific promoters for plant transformation (Potenza et al. 2004). A large T-DNA tagged rice mutant library (TRIM), formed by placing a promoter-less GUS gene next to the right border of T-DNA (Yu et al. 2007), is very useful, but highly expensive and time-consuming for the identification and study of genome-wide promoters. In silico identification of plant root-specific regulatory regions or elements has been demonstrated to be an effective method (Christ et al. 2012). To identify and characterize novel root-specific promoters in rice, we mined the microarray datasets and screened for promoters that conferred root-specific gene expression with a low expression in all other organs under stress or normal conditions, followed by the experimental analysis of the predicted promoter sequences. Finally, the expression levels of seven root-specific genes were evaluated using real-time PCR in different tissues at various growth stages and five genes were further tested for root-specific expression. The activities of the corresponding rRSP1-5 (rice root-specific promoters 1-5) were subsequently examined in transgenic rice using a GUS reporter gene and in transient transformation of Nicotiana benthamiana using dual-luciferase (Dual-LUC) assays. The exclusive root activities of rRSP1, rRSP3, and rRSP5 demonstrated that these promoters may be suitable for driving root-specific expression in transgenic plants and provided useful alternatives for root-specific transformation of rice and other cereals. This research will provide a useful resource for future genetic engineering and breeding.
Table 1 Information on selected root-specific genes and their expression signal in the roots of three rice varieties under various stress treatments1)
1989). Also, the rolD promoter (Leach and Aoyagi 1991), derived from the Ri (root-inducing) plasmid of Agrobacterium rhizogenes, has been used for root-specific expression of the glutamine synthetase gene (Fei et al. 2003) and a high-affinity nitrate transporter gene (Fraisier et al. 2000). Its activity was much stronger than the CaMV 35S Domain A region, especially in the root tips, according to the GUS
DK151/ Tillering stage
DK151/Panicle elongation stage CK Drought 2 465.93 2 104.67 1 859.19 489.07 128.82 111.22 507.43 525.19 2 667.84 3 113.04 318.64 201.48 280.29 391.97
important regulatory element used for root-specific expression (Benfey and Chua
Gene name
2
3
HUANG Li-yu et al. Journal of Integrative Agriculture 2015, 14(1): 1–10
PLACE (plant cis-acting regulatory DNA elements, Higo et al. 1999). Three novel conserved motifs were identified (Appendix A) and several known root-specific cis-element were overrepresented, such as ROOTMOTIFTAPOX1, OSE1ROOTNODULE, RHERPATEXPA7, SURECOREATSULTR11 (Appendix B), as well as many other elements.
Os07g0646800, Os05g0148900, Os04g0423800, Os06g0500700, and Os03g0185700, renamed RSG1-7, respectively, in Table 1) were observed in both young and mature roots, but there was no or low expression in seedlings and mature leaves (Fig. 1). These genes also specifically and stably expressed in roots under PEG, salt and cold conditions (Fig. 2).
2.3. Root-specific activity validation in rRSP1-5:: -GUS transgenic plant
2.2. In silico identification root-specific motifs of regulatory regions
To investigate the spatial and developmental regulation of rRSP1-5 (rRSP6 and rRSP7 DNA fragment couldn’t be obtained for some unknown reasons), we used the β-glucuronidase (GUS) reporter gene and fused it the approximately 2500-bp regions upstream of the translation start sites of RSG1-5, followed by introducing the constructs into rice for histochemical analysis (Fig. 3-A). Three independent T1 transgenic lines containing each of rRSP1-5::GUS were analyzed for GUS activities in the seedlings, roots, stems, leaves, glumes and seeds. Strong GUS activity was detected at 21 days after germination (DAG) in the primary root, lateral root, and root elongation region of rRSP1::GUS, and rRSP5::GUS transgenic rice, and weakly in rRSP3::GUS transgenic plants. Weak GUS activity was detected in the stems of rRSP2::GUS and in the leaves of rRSP4::GUS (Figs. 3-B and 4-A). Furthermore, no GUS staining was observed in root hairs, root tips, glumes and seeds for any of the rRSP1-5::GUS transgenic plants, but it was for the control A2P::GUS transgenic plants
Promoters play key roles in conferring temporal, spatial, chemical, developmental, or environmental regulation of gene expression. They usually contain essential cis-acting elements that interact with transcription factors to regulate transcription upon perception of upstream signals. To identify cis-elements rather than regulatory genomic regions conferring root-specific expression on the seven genes, we analyzed the expression of neighboring genes that might affect the expression pattern of the root-specific genes. The results identified a control region that was able to influence the expression of several genes, but not RSG1-7, based on the Rice Oligonucleotide Array Database (ROAD), a public resource for gene co-expression analysis. To define these rice root-specific promoters (rRSP1-7) containing conserved motifs or cis-elements, approximately 2 500-bp upstream of the translation starting point (ATG) was analyzed using MEME (multiple EM for motif elicitation, Bailey et al. 2009), the Perl procedure of Tomodel by
0.002 0.001 0.000
1
2
3
4 F
0.050
RSG5
0.040 0.030 0.020 0.010 0.000
1
2
3
4
0.40 0.30 0.20 0.10 0.00
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2
3
4 G
0.07
RSG6
0.06 0.05 0.04 0.03 0.02 0.01 0.00
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2
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4
0.030
Relative expression level
0.003
RSG2
D
Relative expression level
0.004
Relative expression level
RSG1
0.005
C
0.50
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Relative expression level
E
B
0.006
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Relative expression level
A
RSG3
0.025 0.020 0.015 0.010 0.005 0.000
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RSG4
0.005 0.004 0.003 0.002 0.001 0.000
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2
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H
0.0025
RSG7
25 c 23 c 26 c 26 c 25 c 27 c 26 c 22 c
0.0020 0.0015 0.0010 0.0005 0.0000
0.006
1
2
3
1
2
3
4
RGS1 RGS2 RGS3 RGS4 RGS5 RGS6 RGS7 Actin
4
Fig. 1 Expression mode of selected root-specific genes (RSG) in different tissues. Analysis of RSG1-7 expression in mature and young leaves (1 and 2), mature and young roots (3 and 4) by qRT-PCR (A–G) and RT-PCR (H). Actin 1 was used as a reference. Bars are means±SD. The same as below.
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HUANG Li-yu et al. Journal of Integrative Agriculture 2015, 14(1): 1–10
0.004 0.003 0.002 0.001 0.000
1
2
3
4
0.08 0.06 0.04 0.02 0.00
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3
0.015 0.010 0.005 0.000
1
2
3
4
0.0015 0.0010 0.0005 0.0000
1
2
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4
G 0.007
RSG6
0.006 0.005 0.004 0.003 0.002 0.001 0.000
1
2
3
4
0.012
RSG4
0.010 0.008 0.006 0.004 0.002 0.000
1
2
2
3
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4
H
Relative expression level
RSG5
0.020
RSG3
0.0020
4
F 0.025
Relative expression level
Relative expression level
E
RSG2
D
0.0025
Relative expression level
RSG1
C
0.10
Relative expression level
B
0.005
Relative expression level
Relative expression level
A
0.0020 0.0018 0.0016 0.0014 0.0012 0.0010 0.0008 0.0006 0.0004 0.0002 0.0000
RSG7
1
2
3
25 c 23 c 26 c 26 c 25 c 27 c 26 c 22 c
4
1
4
RGS1 RGS2 RGS3 RGS4 RGS5 RGS6 RGS7 Actin
Fig. 2 Expression mode of selected root-specific genes under various stresses. Analysis of RSG1-7 expression in roots under normal, PEG, salt and cold treatment (1, 2, 3, 4) by qRT-PCR (A–G) and RT-PCR (H). Actin 1 was used as a reference.
ATG
A2P/RSPs
A Pacl RB
ccdB
C
Ascl
Cmr attR1
GUS
attR2
gus
nos T
Hygr
LB
pMDC162 100 μm
rRSP4::GUS rRSP5::GUS
rRSP3::GUS
rRSP1::GUS rRSP2::GUS
A2P::GUS
B
Fig. 3 Diagrams showing tissue-specific GUS staining in transgenic rice plants. A, schematic diagram of A2P/rRSP1-5::GUS. The entire expression cassette is flanked by the left border (LB) and right border (RB) sequences in the pMDC162 binary vector, which uses an Hpt gene as the selectable maker gene. B, GUS staining results of root, stem, leaves, glume and seed from A2P/ rRSP1-5::GUS transgenic rice. C, cross section of rRSP5::GUS transgenic rice roots.
HUANG Li-yu et al. Journal of Integrative Agriculture 2015, 14(1): 1–10
(Figs. 3-B and 4-A). A2P was previously characterized as a strong constitutive promoter. Additionally, GUS staining was detected in the columella of primary roots and lateral roots of rRSP5::GUS transgenic rice (Fig. 3-C). Taken together, the staining results demonstrated that rRSP1, rRSP3, and rRSP5 had root-specific activity patterns.
2.4. Analysis of rRSP1-5 activity at the RNA level by qRT-PCR of GUS expression To quantify the activities of rRSP1-5, qRT-PCR was used to detect the expression patterns and levels for each promoter in the transgenic plants; the GUS mRNA levels of three independent T1 A2P/rRSP1-5::GUS transgenic lines were measured. Total RNA was extracted from roots at 21 and 90 DAG. rRSP1-5 promoter activities were analyzed in comparison with A2P. As shown in Figs. 3-B and 4, GUS mRNA accumulation in samples from rRSP1, rRSP3, and rRSP5 transgenic lines occurred specifically in the roots, but not in other tissues, demonstrating that rRSP1, rRSP3, and rRSP5 were activated in a root-specific pattern. The GUS transcript levels driven by the rRSP1, rRSP3, and rRSP5 were different: 0.3-, 0.1- and 0.6-fold viruses A2P in the roots, respectively. GUS mRNA levels in rRSP2 and rRSP4 transgenic lines were 0.8- and 0.2-fold of the A2P level in the roots, but detectable in both shoots and roots. Although the rRSP1-5 activities varied between different transgenic lines, highly root-specific expression patterns were observed. Overall, our results demonstrated that rRSP1, rRSP3 and rRSP5 were specifically activated in roots.
A
5
2.5. rRSP1-5 have different tissue activities in Nicotiana benthamiana leaves To detect the activities of rRSP1-5 in other species, a Dual-LUC assay was employed to analyze their transient activities. rRSP1-5 were fused with the firefly luciferase gene (LUC) to analyze their transient activities and the REN luciferase gene (REN) under the control of the 35S promoter was used as an internal reference to allow for the effects of transformation efficiency (Fig. 5-A). As shown in Fig. 5-B, the A2P::LUC showed the strongest luciferase activity in comparison with rRSP1, rRSP3, rRSP5::LUC, which was consistent with activities observed in rice. However, rRSP1, rRSP3 and rRSP5 with root-specific expression in rice had relative lower activities in N. benthamiana leaves, indicating these promoters are not specific in roots of N. benthamiana.
3. Discussion Tissue-specific promoters control gene expression in a tissue-dependent manner, the use of such promoters for specific expression of exogenous genes in the corresponding tissues is more suited to the nature of rice growth and makes the expression of foreign genes in rice more specific, safe and efficient. Soil nutrient deficiencies and water limitation are the most significant constraints on crop productivity. A genetically engineered root system may be particularly advantageous for aiding the absorption of water and nutrients from deep soil layers, which is a strategy for drought avoidance and nutrient acquisition (Fukai and
US US US US US US ::G ::G ::G ::G ::G G 1 2 3 4 5 : P: SP SP SP SP SP B A2 rR rR rR rR rR
21 DAG 90 DAG
20.00 15.00 10.00 5.00 0.00
rR
A2 P
::G SP US 1 rR ::G SP US 2: :G rR SP US 3: :G rR SP US 4: :G rR SP US 5: :G US
Relative expression level
25.00
Fig. 4 Promoter activity of rRSP1-5. A, GUS staining in the whole plants of the A2P/rRSP1-5::GUS transgenic seedlings. B, analysis of rRSP1-5 activity on the RNA level of the GUS gene by qRT-PCR.
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HUANG Li-yu et al. Journal of Integrative Agriculture 2015, 14(1): 1–10
A
A2P/rRSP1-5
LB
RB
2×35S 35S T
REN B
LUC
35S T
0.6
LUC/REN
0.5 0.4 0.3 0.2 0.1 0.0
A2P
rRSP1
rRSP2
rRSP3
rRSP4
rRSP5
Fig. 5 rRSP1–5 have different tissue activities in Nicotiana benthamiana leaves. A, schematic diagram of A2P::LUC. The entire expression cassette is flanked by the left border (LB) and right border (RB) sequences in the pGreen II-0800 binary vector. 35S::REN was used as a reference. B, Transient activity analysis in N. benthamiana.
Cooper 1995; Yue et al. 2006). Traditional breeding methods frequently fail to exploit traits controlled by many genes; however, the application of genetically engineered plants in crop breeding could help researchers gain control of those traits. Root-specific promoters are of particular interest in these applications. Although several root-specific promoters have been isolated and functionally characterized from bacteria, viruses, and plants, few are suitable for monocotyledonous plants. It was reported that the non-plant promoters including rolD promoter and the CaMV 35S promoter domain A direct high levels of root-specific transgene expression in dicotyledonous plants (Benfey and Chua 1990; Leach and Aoyagi 1991; Elmayan and Tepfer 1995). The tobacco TobRB7 promoter is expressed highly and specifically in roots. Other dicot promoters have been used for monocot transformation, but their activity tends to be lower in the monocot (Wilmink et al. 1995). Monocot promoters of the rice RCc3, Os03g01700
and Os02g37190 genes have produced high levels of gene expression in rice (Jeong et al. 2010; Li et al. 2013), but there is a lack of evidence concerning their effectiveness in other species. Hence root-specific promoters showing moderate activity need to be identified urgently. Our results demonstrated the root specificity and expression levels of the promoters for five rice genes, RSG1-5, as shown in Figs. 1 and 2 and Table 1. Although rRSP1-5::GUS expression varied with the root tissues and stages, rRSP1, rRSP3 and rRSP5 were clearly active in the roots (Figs. 3-B and 4). Their expression patterns suggested that rRSP1, rRSP3 and rRSP5 promoters were active in various root cell types, which is distinct from other previously identified root-specific promoters. For example, the rolD promoter exhibited widespread activity in mature root tissues, but its root-specific activities are completely absent in young roots (Leach and Aoyagi 1991; Elmayanand and Tepfer 1995). Domain A of the CaMV 35S promoter is mainly
Table 2 Activity of root-specific promoters tested in rice Vector A2P::GUS rRSP1::GUS rRSP2::GUS rRSP3::GUS rRSP4::GUS rRSP5::GUS
Size of the region Expression tested (bp) pattern 1 405 All tissues 2 171 Primary root, lateral root 2 336 Primary root, lateral root 2 311 Part of the primary root and lateral root 1 706 Primary root, lateral root 2 236 Columella of primary root and lateral root
Strength of expression in root Strong Intermediate Strong Weak Intermediate Strong
Expression in non-root organs Yes No Weak in stems and leaves No Weak in stems and leaves No
HUANG Li-yu et al. Journal of Integrative Agriculture 2015, 14(1): 1–10
active in root tips (Elmayan and Tepfer 1995), but at a much lower level than the rolD promoter. The tobacco TobRB7 promoter exhibits root-specific activity as early as 2 DAG in tobacco transgenic plants, and the highest levels of activity of this promoter are detected in the meristem and central cylinder (Yamamoto et al. 1991). The expression patterns of Os03g01700 and Os02g37190 are similar to those of the TobRB7 and rolD promoters, but the former showed more constitutive activity in the root than the latter promoters. The distribution of GUS staining in the transgenic rice plants for rRSP1, rRSP3 and rRSP5 revealed moderate and varied activities of these root-specific promoters. Peculiarly, GUS staining was only visible in the columella of primary and lateral roots of rRSP5::GUS transgenic rice. However, no GUS activity was observed in the stem, leaves and panicle, or in the seeds, which guarantees the food safety in exogenous gene transformation breeding. rRSP2 and rRSP4, which showed low transcriptional activity in certain over-ground tissues, can be potentially used for the transgenic breeding of some authigenic genes. The analysis of conserved motifs or cis-elements conferring root-specific expression indicated that three novel motifs are overrepresented in their promoters (Appendix A). As shown in Appendix B, a large range of previously characterized elements were common in these promoters, and a variety of tissue-regulatory elements in the promoter regions were also identified, such as ROOTMOTIFTAPOX1, CACTFTPPCA1, CAATBOX1, RHERPATEXPA7, SURECOREATSULTR11 and GTGANTG10. rRSP1 contains at least 20 ROOTMOTIFTAPOX1 elements and 35 CACTFTPPCA1 elements. rRSP3 has at least nine ROOTMOTIFTAPOX1 elements, 27 CAATBOX1 elements and 34 CACTFTPPCA1. Similarly, rRSP5 contains at least 10 OSE1/2ROOTNODULE elements and 35 CACTFTPPCA1 elements (Appendix B). ROOTMOTIFTAPOX1 and RSE (root-specific element) (Benfey and Chua 1989; Keller and Baumgartner 1991; Elmayan and Tepfer 1995), and CACTFTPPCA1 is a key component of mesophyll expression module 1 (Mem 1), which is found in the cis-regulatory element in the distal region of the ppcA1 gene of the C4 dicot Flaveria trinervia (Gowik et al. 2004). Various elements distributed throughout these promoters demonstrate that synergistic interactions occur among cis-elements (Benfeyand Chua 1989). Furthermore, the diversity of cis-acting elements in the promoters may be responsible for their root-specific expression and varied expression levels. Transient activity analysis in N. benthamiana demonstrated that rRSP1-5 have similar expression modes to those in rice (Figs. 4 and 5-B). We hypothesized that many homologous transcriptional factors cooperate to regulate their expression among species. However, further study is required to elucidate the mechanism of their root-specific
7
expression. Although the expression patterns of RSG1-7 were similar in the roots under stress conditions, there were significant differences. The characterization of rRSP1, rRSP3, and rRSP5 indicated that their downstream genes are expressed in root cell types of transgenic rice and supports their potential for targeting a wide spectrum of root cells. In addition, the ability to drive root-specific expression make rRSPs useful for improving nutrient and water uptake, root growth and drought tolerance.
4. Conclusion In this study, micro-array and RT-PCR led to the identification of seven root-specific expression genes in rice. The isolation and characterization of upstream promoter region of five selected genes (rRSP1-5) and A2P (promoter of OsAct2) revealed that rRSP1, rRSP3, and rRSP5 are specifically important with respect to the root-specific activity. Further, rRSP1, rRSP3, and rRSP5 were implicated to differently contribute to the root activity in various specifies. Those three promoters have the potential to be applied to the root-specific enhancement of target gene(s).
5. Materials and methods 5.1. Plant materials Rice (Oryza sativa L. cv. Nipponbare) was used to examine the expression of endogenous genes, to isolate gene promoters, and to perform plant transformations. Disinfected Nipponbare seeds were germinated at 37°C for 3 d. Uniform seeds were selected and grown hydroponically in a PCR plate containing Yoshida nutrient solution (Yoshida et al. 1976) in a well-controlled greenhouse. Seedlings at 21 DAG were exposed to low-temperature (4°C), salinity (150 mmol L–1 NaCl), or drought (20% PEG-6000, w/v) conditions. Root and leaf samples were taken from each plant at 0 and 24 h after treatment, immediately frozen in liquid nitrogen and stored at –70°C. Mature leaves and roots were sampled at the booting stage (90 DAG) from plants growing in the field and processed using the above method.
5.2. Identification of root-specific genes from the microarray data Signal expression data files were obtained from datasets (Wang et al. 2011). The two datasets were independently preprocessed following the stipulations which expressed in roots but not in other tissues and stable under various stresses (fold change
