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J. Crop Sci. Biotech. 2009 (March) 12 (1) : 25 ~ 30 DOI No. 10.1007/s12892-008-0054-8 RESEARCH ARTICLE
In Silico Identification of Cis-Regulatory Elements of Phosphate Transporter Genes in Rice (Oryza sativa L.) Marcelinus Rocky Hatorangan*, Erwin Sentausa, Grace Yasmein Wijaya Bioinformatics Laboratory, Faculty of Technobiology, Atma Jaya Catholic University of Indonesia, Jalan Jenderal Sudirman 51, Jakarta 12930 Received: September 30, 2008 / Accepted: March 23, 2009 Ⓒ Korean Society of Crop Science and Springer 2009
Abstract Promoter sequences of 13 Phosphate Transporter genes of Oryza sativa L. (OsPTs) have been analyzed in silico to identify their cis-regulatory elements (CREs). The DNA sequences of these OsPT genes were mined from NCBI gene databases. MEGABLAST program was used to align these sequences to Rice Genome Database in obtaining their complete sequences. The upstream region (-1 to -400) of the complete gene sequences were analyzed using SIGNALSCAN program provided by PLACE database of cis-regulatory element motives. From this procedure, 153 types of CRE were identified. Four of these CREs were found on all of the OsPT genes: ARR1AT, CAATBOX1, CACTFTPPCA1, and DOFCOREZM. Among these CREs, CACTFTPPCA1 was found with 3 to 15 duplications in each cis-regulatory sequence. In addition, each OsPT gene has typical CREs that can only be found in the respective genes. The total number of these typical CREs is 54, and one of them was a binding site for a bHLH-like protein, CACGTGMOTIF or the G-box. Moreover, several E-boxes which also functioned as a binding site for a bHLH-like protein were identified in all OsPTs except in OsPT1, OsPT6, and OsPT8. There were significant correlations (p < 0.05) between mRNA levels of OsPT1 to OsPT11 in rice root with or without inoculation of Glomus intradices reported by Paszkowskidagger et al. (2002) and the duplication numbers of ARR1AT, CAATBOX1, CACTFTPPCA1, CURECORECR, and WRKY71OS. Key words: phosphate transporter, cis-regulatory elements, rice
Introduction Phosphorus (P) is an essential macronutrient for plants. The availability of P in the environment supports the growth, development, and reproduction of plants. This element is absorbed by plants in its inorganic form and transported inside the cell as anion (Schachtman et al. 1998). Phosphorus intake by plant root is limited because of its ability to form ferric phosphate as well as aluminum phosphate on acidic land and to form calcium phosphate as well as magnesium phosphate on basic land. These forms of compound make it harder for phosphorus to enter plant roots (Bar-Yosef 1991). To solve this problem, soil enrichment using phosphate-based fertilizers is regularly conducted. However, this effort can raise Marcelinus Rocky Hatorangan ( Email:
[email protected] Tel: +62-812-87287-19
)
The Korean Society of Crop Science
another problem especially when the phosphate dissolves by water and drains into the aquatic ecosystem. Moreover, the phosphate fertilizers are relatively expensive. Phosphate uptake in plants correlates with the membrane transport system which is mediated by proteins of phosphate transporters. Research conducted by Liu et al. (1998) showed that inorganic phosphate (Pi) available in soil influences the expression of some phosphate transporter genes and enhances the phosphate uptake at a current concentration level. According to Okumura et al. (1998), based on their Km values phosphate transporters were divided into two groups, the high affinity and the low affinity phosphate transporters. High affinity phosphate transporters have the ability to deplete phosphate from soil in the µM range concentration, while the low affinity can do their action only in the mM concentration range. Paszkowskidagger et al. (2002) identified 13 homologue rice gene sequences that
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Cis-Regulatory Elements of Phosphate Transporter Genes in Rice
were similar to high affinity Pi transporters. These genes were named Oryza sativa Phosphate Transporter (OsPT) 1 to 13. Yi et al. (2005) found that OsPTF1, a basic helix-loop-helix transcription factor which commonly bind to the consensus sequence on the upstream region of a gene called the E-box (5'CANNTG-3'), is involved in Pi transport. Based on this finding, interaction between the transcription factors with their binding site becomes important. Hence, the aim of this study is to identify other cis-regulatory elements (CREs) in the -1 to -400 region upstream from the commonly known phosphate transporter genes (OsPT1-13) which are involved in phosphate uptake by cells in rice (Oryza sativa L.).
Table 1. Rice Chromosome and Phosphate Transporter Genes. Location* Length* OsPT Chromosome (Base Pair) (Base Pair) 1 3
4
6 8 10
Materials and Methods The DNA sequences of the OsPTs based on study conducted by Paszkowskidagger et al. (2002) were mined from National Center for Biotechnology Information (NCBI) gene databases. OsPTs sequences retrieved from NCBI were limited to gene sequences only. MEGABLAST (Zhang et al. 2000) program was used to align these sequences to Oryza sativa (japonica-cultivar group) genome (reference only). The expectation number was set to 0.01 with default filter. Analysis of -1 to -400 OsPTs upstream gene region (promoter) was conducted by using the SIGNALSCAN program provided by Plant cis-acting Regulatory DNA Elements (PLACE), database of cis-regulatory element motif (Higo et al. 1999; Prestridge et al. 1991). Cis-regulatory elements (CRE) for further analysis were selected conducted based on their duplication and distribution number. The selected CREs were mapped manually based on their location in the upstream promoter region. The Spearman Rank Correlation method was conducted in order to compare the expression level of OsPTs in rice root and the duplication number of each CRE. The compared OsPTs expression level data were based Paszkowskidagger et al. (2002) experiment data.
11 1 2 7 12 4 5 13 9 10 6 3 8
26,717,152 2,789,180 2,797,023 1,991,798 2,785,498 5,811,777 5,775,714 5,829,517 12,521,672 12,533,979 28,236,489 15,513,193 15,521,741
-
26,720,711 2,791,763 2,799,609 1,994,378 2,788,123 5,814,393 5,778,421 5,833,519 12,525,999 12,538,146 28,239,093 15,515,668 15,524,366
3559 2583 2586 2580 2625 2616 2707 4002 4327 4167 2604 2475 2625
* including the -400 region
Fig. 1. The OsPTs locations in rice chromosomes. Except for OsPT3 with 99% identity hit score, other OsPTs locations were retrieved based on 100% identity hit score when OsPTs sequences were aligned with the rice genome using MEGABLAST.
Results
AAAG-3'). The duplication numbers of those CREs are represented in Figure 2. The complete mapping for ARR1AT, CAATBOX1, CACTFTPPCA1, and DOFCOREZM location in each OsPT is shown in Figure 3. Among these 4 CREs, the most abundant CRE is CACTFTPPCA1 with 90 duplications in all OsPT genes with 3 to 15 duplications in each OsPT gene. The CREs seem dispers-
The MEGABLAST analysis in rice genome resulted in 13 similar sequences to OsPT1 through OsPT13. Those sequences were found in six different rice chromosomes as represented in Table 1. OsPT1, OsPT2, and OsPT12 were located at the same chromosome, and the locations were close to each other. The same condition was also observed with OsPT4, OsPT5, and OsPT13 at chromosome 4, and OsPT3 and OsPT8 at chromosome 10. The exact location of these OsPTs in each chromosome is presented in Figure 1. The analysis of the -1 to -400 promoter region of rice OsPTs using SIGNALSCAN resulted in 153 types of cis-regulatory elements (CREs). Four of these CREs were found in all of OsPTs, which were ARR1AT (5'-NGATT-3'), CAATBOX1 (5'-CAAT3'), CACTFTPPCA1 (5'-YACT-3'), and DOFCOREZM (5'-
Fig. 2. Numbers of duplication of ARR1AT, CAATBOX1, CACTFTPPCA1, and DOFCOREZM in each OsPT.
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edly distributed along the OsPTs promoter. From this observation, no typical patterns were found. Other common CREs (found in 80% or more OsPTs) are CURECORECR, GATABOX, GTGANTG10, POLLEN1 LELAT52, and WRKY71OS. Beside these CREs, there are several other CREs which were only found in specific OsPTs which are presented in Table 2. Except for OsPT10 and OsPT12, other OsPTs have typical CREs that could be found only in these respective genes. The total number of these typical CREs was 54. Some publications about CREs duplication in yeast (Papp et al. 2003) and promoter duplication in tombusviruses (Panavas et al. 2003) indicate that the duplication numbers of CREs or promoter might have a certain role in leveling the mRNA concentration or gene expression. In this research, similar correlation seemed to exist between the OsPTs mRNA level in rice root with or without inoculation of Glomus intradices reported by Paszkowskidagger et al. (2002) and the duplication number of ARR1AT, CAATBOX1, CACTFTPPCA1, CURECORECR and WRKY71OS (Fig 4). Table 3 shows a complete analysis of General CREs (found in more than 80% OsPTs). Based on Spearman Rank Correlation Coefficient statistic analysis using the GENSTAT Discovery
Edition 3 Release 7.2 program, significant positive correlations (p ≤ 0.05) between ARR1AT, CAATBOX1 duplication numbers and OsPT1 to OsPT11 mRNA levels in Glomus intradices inoculated rice root were found. Another significant positive correlation (p ≤ 0.05) was also found between CACTFTPPCA1, CURECORECR duplication numbers and mock inoculated rice Table 2. Typical CREs found in -1 to -400 promoter region of rice OsPTs. Duplication OsPT No. Typical CRE Signal Sequence Number
1
2
3
4
5
6
7
8 9 10 11
12 Fig. 3. Complete CREs map of CREs found in all OsPT and EBOXBNNAPA positions.
13
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
AMYBOX2 E2FBNTRNR IBOXCORENT SP8BFIBSP8AIB TATCCAYMOTIFOSRAMY3D WRECSAA01 ACGTTBOX PYRIMIDINEBOXHVEPB1 SURE1STPAT21 ACGTOSGLUB1 CCA1ATLHCB1 CIACADIANLELHC GCN4OSGLUB1 LTRE1HVBLT49 MYBATRD22 POLASIG2 POLLEN2LELAT52 SEF3MOTIFGM ABREATCONSENSUS ACGTABREMOTIFA2OSEM BOXIIPCCHS CACGTGMOTIF EMBP1TAEM IRO2OS AACACOREOSGLUB1 RYREPEATBNNAPA RYREPEATGMGY2 RYREPEATLEGUMINBOX RYREPEATVFLEB4 SORLIP4AT SPHCOREZMC1 ANAERO3CONSENSUS MARTBOX NTBBF1ARROLB ARE1 CTRMCAMV35S GAGA8HVBKN3 GAGAGMGSA1 SORLIP5AT WBBOXPCWRKY1 OCTAMOTIF2 SEF1MOTIF SORLIP2AT CMSRE1IBSPOA SREATMSD LECPLEACS2 SORLREP3AT XYLAT AGCBOXNPGLB HBOXCONSENSUSPVCHS HBOXPVCHS15 PREATPRODH REBETALGLHCB21 GT1MOTIFPSRBCS
TATCCAT GCGGCAAA GATAAGR ACTGTGTA TATCCAY AAWGTATCSA AACGTT TTTTTTCC AATAGAAAA GTACGTG AAMAATCT CAANNNNATC TGAGTCA CCGAAA CTAACCA AATTAAA TCCACCATA AACCCA YACGTGGC ACGTGKC ACGTGGC CACGTG CACGTGGC CACGTGG AACAAAC CATGCA CATGCAT CATGCAY CATGCATG GTATGATGG TCCATGCAT TCATCAC TTWTWTTWTT ACTTTA RGTGACNNNGC TCTCTCTCT GAGAGAGAGAGAGAGA GAGAGAGAGAGAGAGAGA GAGTGAG TTTGACY CGCGGCAT ATATTTAWW GGGCC TGGACGG TTATCC TAAAATAT TGTATATAT ACAAAGAA AGCCGCC CCTACCNNNNNNNCT CCTACCNNNNNNNCTNNNNA ACTCAT CGGATA KWGTGRWAAWRW
1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 10 10 10 8 1 1 1 1 2 1 6 3 2 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1
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Cis-Regulatory Elements of Phosphate Transporter Genes in Rice
root (without inoculation). WRKY71OS has a negative correlation (p ≤ 0.05) with OsPT1 to OsPT11 mRNA levels in inoculated or mock inoculated rice root.
Discussion The alignment between the sequences provided by the gene database compared to the genomic sequences resulted in 100% identity hit for each OsPT, except OsPT3 which yielded 99% identity hit. Despite the previous alignment results, the -1 to 400 upstream region of the OsPT1-13 was retrieved.
Fig. 4. The Pi Transporter mRNA levels in Glomus intradices inoculated rice roots (a) and mock inoculated rice root (b) data provided by Paszkowskidagger et al. (2002). Correlation tendency between ARR1AT, CAATBOX1, WRKY71OS, and Pi Transporter mRNA levels in Glomus intradices inoculated rice roots (c) and correlation tendency between CACTFTPPCA1, CURECORECR, WRKY71OS, and mock inoculated (without inoculation) rice root were observed as positive correlation tendency while WRKY71OS has negative tendency.
Table 3. Correlation coefficient between CREs duplication numbers and OsPT1 - OsPT11 mRNA levels in rice root CRE ARR1AT CAATBOX1 CACTFTPPCA1 CURECORECR GATABOX GTGANTG10 POLLEN1LELAT52 WRKY71OS
Glomus intradices inoculated
Mock inoculation
0.782S 0.746S 0.041 0.15 -0.471 -0.125 -0.014 -0.561S
0.468 0.236 0.618S 0.664S 0.125 -0.334 0.505 -0.616S
s: show a significant correlation (p ≤ 0.05)
The presence of EBOXBNNAPA (5'-CANNTG-3') or the Ebox in all OsPT genes except OsPT1, OsPT6, and OsPT8 suggested that those genes might be regulated by bHLH-like proteins. However, the microarray analysis data provided by Yi et al. 2005 shown that none of OsPT genes were regulated by OsPTF1 in transgenic rice which over expressing OsPTF1. On the other hand, according to Atchley and Fitch (1997) and Buck and Atchley (2003), bHLH-like protein in plants binds to more specific E-box called the G-box (5'-CACGTG-3'). In this research, a CACGTGMOTIF or the G-box was located palindromically at -213 promoter region of OsPT4 but not found at the other OsPTs. Laboratory research must be conducted to determine the correct binding site for OsPTF1 besides the E-box or G-box. ARR1AT (5'-NGATT-3') is a binding element for cytokinin response regulator ARR1 which is found in Arabidopsis (Oka et al. 2002; Sakai et al. 2000). It is also found in the promoter of rice non-symbiotic haemoglobin-2 (NSHB) gene (Ross et al. 2004). Cytokinin is well known for its role in regulating phosphate uptake. In Arabidopsis thaliana, external auxin and cytokinin suppress the expression of Arabidopsis thaliana phosphate transporter, AtPT1 (Varadarajan 2003). In addition to that, another research study found that ARR1 that bound to ARR1AT, directly activated the cytokinin response genes (Taniguchi et al. 2007). The latest finding suggests that in rice, cytokinin might have different roles compared in rice and Arabidobsis particularly in regulating transporter genes. These results were confirmed by statistic analysis using the Spearman Rank Correlation Coefficient (GENSTAT Discovery Edition 3 Release 7.2) which showed significant correlations (p < 0.05) between the mRNA levels in Glomus inoculated rice root and the duplication number of ARR1AT. This significant correlation showed a tendency for ARR1AT to up regulate those genes expression in the presence of Glomus. CAATBOX1 (5'-CAAT-3') is a sequence responsible for the tissue specific promoter activity of a pea legumin gene (Shirsat et al. 1989). Legumin is a major component of pea seed storage vacuoles (Stoger et al. 2001). The relationship between legumin, and phosphate uptake is not well known. CACTFTPPCA1 (5'-YACT-3') is a cis-regulatory element found in the C4 plant Flaveria trinervia, and part of the promoter of the C4 phosphoenolpyruvate carboxylase gene (Gowik et al. 2004). This CRE is a key component of Mem1 (mesophyll expression module 1) protein in the same plant. CACTFTPPCA1 might have a different role in C3 plants like rice. Phosphoenolpyruvate carboxylase is well known for its role in succulent or C4 plants' photosynthesis in adding CO2 to phosphoenolpyruvate (PEP) (Mazelis and Vennesland 1957). In wheat (Triticum aestivum), the combination of phosphate and
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copper in soil had several effects in increasing the photosynthesis rate (Javadi et al. 1991). CURECORECR (5'- GTAC -3') is a nucleotide sequence essential for copper responsive genes (Cyc6 and Cpx1). This gene is found in Chlamydomonas reinhardtii (Quinn et al. 2000). However, the existence of similar gene sequences has not yet been identified in rice. Another CRE, WRKY71OS (5'-TGAC-3'), is a binding site for WRKY71, a transcriptional repressor of the gibberellin signaling pathway (Zhang et al. 2004). Interaction between gibberellin and phosphate uptake is not well known. However, research conducted by Koehler et al. (2004) on barley aleurone cells showed that gibberellins might be involved in phosphate uptake by controlling Pi incorporation into phospholipids. Different significant correlation between the CREs and the mRNA levels in Glomus inoculated or mock inoculated rice root might be due to the basic function of the CRE itself. Based on the literature, both CACTFTPPCA1 and CURECORECR are related to the photosynthesis process. Meanwhile, the ARR1AT, CAATBOX1, and WRKY71OS have some relation with plant hormone response. Research conducted by Danneberg et al. (1993) showed that Glomus inoculation in maize (Zea mays) affects the phytohormone balances. This might explain why there are some correlations between the ARR1AT, CAATBOX1, and WRKY71OS with the OsPTs mRNA levels in Glomus inoculated rice root. The importance of CREs in regulating gene expression is already well known. Cai et al. 2007 made an experiment using PD540, a tissue specific promoter from rice. They found that truncated PD540 and several of its fragments had different roles in regulating gene expression. LPSE1, one of the CREs found in PD540 fragments, activate gene expression in leaf and young panicles. Another CRE found in the same promoter, LPSRE2, suppressed gene expression in leaf, root, young panicles, and stem. Besides controlling tissue-specific gene expression, CREs are also found to control condition-dependent gene expression. The Ibox, a CRE in the upstream of rbcS, cab, and nia genes found in plants, is involved in the regulation of transcription by light and the circadian clock (Borello et al. 1993). Other CRE called the Heat Shock Element (HSE) is responsible for heat shock induction of gene expression and contributed partially to the induction of gene expression by oxidative stress (Storozhenko et al. 1998). The correlations between CREs duplication numbers and OsPTs expressions level found in this research suggest that duplication numbers of specific CREs might have a typical role in increasing or suppressing a certain gene expression. In the future, these results might open a possibility to alter phosphate utilization in rice breeding by adding or decreasing specific CREs' duplication numbers in OsPTs upstream region.
The mRNA levels for OsPT12 and OsPT13 data are not included in this research. Further data on OsPT12 and OsPT13 mRNA levels are necessary in order to yield more specific results. The mRNA levels in specific plants organ or tissue are also needed to make further comparisons.
Conclusion Analysis of -1 to -400 promoter region of OsPT genes using the SIGNALSCAN provided by PLACE yielded 153 CREs. From 153 CREs in this research, there are several putative CREs which might be involved in the regulation of OsPTs expression. CACTFTPPCA1 is the most abundant of all CREs found in OsPT Genes. Other CREs, such as CAATBOX1, DOFCOREZM, and ARR1AT were also found in all the OsPT genes. Moreover, binding sites for bHLH-like proteins (E-box and G-box) that might regulate these phosphate transporter genes were also found in some OsPT genes. Correlations exist between the OsPT1 to OsPT11 mRNA concentration in rice root with or without inoculation of Glomus intradices and the ARR1AT, CAATBOX1, CACTFTPPCA1, CURECORECR, and WRKY71OS duplication numbers. ARR1AT, CAATBOX1, CACTFTPPCA1, and CURECORECR seemed to up regulate the expression of OsPT genes while other CRE called WRKY71OS down regulate them. In the next step, laboratory research must be conducted to verify these results.
Acknowledgement We want to thank Dr. Rory Hutagalung for his support and suggestions in the statistical section.
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