International Research Journal of Plant Science (ISSN: 2141-5447) Vol. 4(5) pp. 109-116, May, 2013 Available online http://www.interesjournals.org/IRJPS Copyright © 2013 International Research Journals Full Length Research Paper Molecular Cloning of Senescence-Related cDNA, OsRab7, from Thai Jasmine Rice (Oryza sativa L. cv. KDML 105) Sugunya Pitakrattananukool1, Supranee Sitthiphrom2, Robert W. Cutler3, Somboon Anuntalabhochai1,4* *1 Department of Biology, Faculty of Science, Chiang Mai University, Muang, Chiang Mai 50200, Thailand. 2 Faculty of Science and Technology, Loei Rajabhat University, Loei, 42000, Thailand. 3 Program of Physics, Department of Science, Edison State College, Ft. Myers, Florida, 33919, USA. 4 * Biotechnology Unit, University of Phayao, Muang, Phayao, 56000, Thailand. *Corresponding Author E-mail: firstname.lastname@example.org Abstract In this work we cloned an OsRab7 DNA sequence encoding a small GTP-binding protein from Thai jasmine rice (Oryza sativa L. cv. KDML 105). All of the characteristic motifs of the small GTP-binding Rab family proteins were present in the OsRab7 sequence. The expression analysis revealed that this cDNA was present in various rice tissues including roots, young leaves, senescing leaves, flowers and seeds. In addition, the cDNA was also up-regulated under the following induced-senescence conditions, abscisic acid (ABA), ethylene, NaCl, dark treatment and wounding, suggesting that OsRab7 acted as a stress-inducible gene involved in the senescence process in Thai jasmine rice. Key words: Small GTP-binding protein, OsRab7, stress-inducible gene, leaf senescence INTRODUCTION The small GTP-binding proteins are known as monomeric G proteins with molecular masses of 20-40 kDa. Several lines of evidence indicate that the small GTP-binding protein existed in eukaryotes and regulated diverse processes in eukaryotic cells including cell proliferation, signal transduction, vesicular transport, cytoskeleton organization and intracellular membrane trafficking. Their molecular function was to switch cycling between the guanosine triphosphate (GTP) activated and guanosine diphosphate (GDP) inactivated state (Bischoff et al., 1999; Ma, 2007). In particular the Rab family contains a large number of proteins which play an important role in various intracellular trafficking pathways in plants (Agarwal et al., 2009). This family is divided into 8 subfamilies designated A-H for each individual class (Vernoud et al., 2003). Most Rab GTPase are approximately 24 kDa in molecular weight containing about 220 amino acids. A total of five Rab subfamily regions (RabSF) and four Rab family regions (RabF) have been described, where these designations correspond to the surface loops involved in proteinprotein interactions. The hypervariable region at the C- terminus called the dicysteinyl prenylation signal is indicated by a ‘CC’ (Pereira-Leal and Seabra, 2001). Several cDNA regions encoding Rab proteins have been cloned from various plant species and their functions have been characterized. For example, the Rha1(Rab5) region was shown to play a role in vascular trafficking in the root tip and stomal guard cells (Anuntalabhochai et al., 1991; Terryn et al., 1993; Sohn et al., 2003). Rab5a was shown to be involved in vesicular membrane transport and also participate in the transport of proglutelin from the Golgi to the PSV. Furthermore, Rab5a is required for the maintenance of the general structural organization of the endomembrane system in developing rice seeds (Fukuda et al., 2011). The transcript expression of the PgRab7 gene isolated from Pennisetum glaucum was found to be differentially up-regulated by such environmental stimuli as cold, dehydration and NaCl and also by the plant hormone IAA. Overexpression of the PgRab7 gene enhanced tolerance to NaCl and mannitol in transgenic tobacco (Agarwal et al., 2008). Moreover, Vanlandingham and Ceresa (2009) reported that RAB7 is required for the 110 Int. Res. J. Plant Sci. transfer of cargo from the late endosome/multivesicular body to the lysosome and for endocytic organelle maintenance. Leaf senescence is the final stage of leaf development and involves programed cell death. The rice senescence process is similar to that of other plants and starts from the lower leaves before extending upward as the plant grows with spreading leaf yellowing. This significant change in cell structure comes from the breakdown of the chloroplast and degradation of various macromolecules. The degraded products are then transferred to sink organs as recaptured nutrients (Lim et al., 2007). From molecular studies during senescence, many genes either have gradually increasing expression levels or are completely switched off. Moreover, many new transcripts were shown to have new functions during senescence. Recently, some members of the Rab genes were reported as being involved in leaf senescence. For example, Price et al. (2008) determined that some Rab subfamily genes were upregulated in senescent of the wallflower petal, and RabG3b was found to be a modulator for cell death progression during the pathogen response and senescence process in Arabidopsis (Kwon et al., 2009). In this work we demonstrate that the OsRab7 gene is related to senescence in Thai jasmine rice and characterize the function and sequence of this member of the small GTP-binding proteins. MATERIALS AND METHODS Plant Preparation Thai jasmine rice (Oryza sativa L. cv. KDML 105) seeds were germinated on agar containing MS media (Murashige and Skoog, 1962) under continuous light at o 25 C. After 10 days of germination, the roots were collected from some rice seedlings and other rice seedlings were then transferred into soil in a greenhouse. The third leaf blades were collected at 20, 30, 40 and 50 days after germination (DAG), in addition to the flowers and seeds at 15 days after flowering (DAF). Plant treatment and Induced-senescence Condition For the plant growth regulator treatments, the third leaf samples were chopped to about 2 to 3 cm in length segments. These chopped rice leaves were then incubated in 3 mM MES buffer at a pH of 5.8 supplemented with 1 mM ethephon and 100 µM of abscisic acid (ABA) under continuous light to examine the effect of these growth regulators. The leaf samples were taken at 0, 3, 6, 12 and 24 h respectively. For the salinity treatments, the chopped rice leaves were incubated in 3 mM MES buffer pH 5.8 supplemented with 100 mM and 200 mM NaCl under continuous light. The leaf samples were taken at 0, 3, and 5 days respectively. For the dark treatment, the chopped leaves were incubated in 3mM MES buffer pH 5.8 and kept in darkness and incubated for 0, 3 and 5 days. For the wounding treatment, the third leaf samples of the rice seedlings at 15 DAG were wounded using a needle stab. Leaf samples were then taken at 0, 3 and 5 days after wounding. Measurement of Chlorophyll Content 2 Chlorophyll contents were measured for 0.6 x 0.6 cm rice leaf samples. The tissues were weighed and ground in liquid nitrogen, and then extracted with 100% methanol. The absorbance was measured at 652.0 nm and 665.2 nm. The chlorophyll concentration per fresh weight was then calculated using the equation described by Porra (2002). chlorophyll (a+b) = 22.12(A652.0) + 2.71(A665.2) Cloning of a Full-length OsRab7 cDNA from Thai Jasmine Rice The partial cDNA sequences encoding the OsRab7 protein were obtained using PCR amplification and total RNA was extracted from the rice leaves at 40 DAG using Trizol reagent (Invitrogen). First-strand cDNA was then synthesized using Superscript III Reverse transcriptase (Invitrogen) following the manufacturer’s instructions. The first-strand cDNA was used as the template for the PCR amplification. The degenerate forward primer was designed to correspond to the highly conserved Nterminal region of the small GTP-binding proteins. The forward primer (OsRabFD), 5’GGNGAYNHNGSNRYNGGNAAR-3’, was constructed to match a sequence coding for the highly conserved 2+ phosphate/Mg binding domain; DTAGQE(RKS). A poly-A oligonucleotide was also used as a reverse primer, oligo(dA)18; 5’-AAAAAAAAAAAAAAAA AA-3’. The amplification conditions were as follows, an initial o denaturation at 94 C for 2 min, followed by 35 cycles at o o 94 C for 45 s, annealing at 50 C for 30 s, extension at 72 o o C for 1 min, and a final extension at 72 C for 10 min. The DNA fragments were ligated into the pTZ57R vector (Fermentas). DNA sequencing was performed using the dideoxynucleotide chain termination method (Sanger et al., 1997) using an automated sequencer (ABI). The fulllength of the OsRab7 cDNA sequences were obtained using by 5’ Full Core Set (Takara) following the manufacturer’s instructions. Pitakrattananukool et al. 111 Semi Quantitative (QPCR) Reaction Reverse Transcriptase PCR Total RNA was extracted from the rice tissues using Trizol reagent and first-strand cDNA was synthesized using a Superscript III Reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Fifteen ng of first-strand cDNA was used as the PCR template. The amplified PCR product sizes were in the 200 - 500 bp range. The QPCR reaction was performed as follows: o initiation for template denaturation for 2 min at 94 C o followed by 26 cycles of denaturation for 30 s at 94 C, o o annealing for 30 s at 62 C and extension for 30 s at 72 C. Rice β-Actin transcripts were used as the internal standards. QPCR Analysis Agarose electrophoresis was performed to visualize the PCR products and photographs were taken. The intensities of the bands were analyzed using Scion Image software (Scion Corporation, Frederick, Maryland) to monitor the relative level of gene expression. The level of transcript expression was presented as the ratio of expression of OsRab7/β-Actin. RESULTS AND DISCUSSION Cloning Of Full-Length cDNAs Encoding Small GTPBinding Rab Family Proteins From Thai Jasmine Rice We attempted to isolate cDNA transcripts encoding small members of the GTP-binding Rab family from Thai jasmine rice. First, the partial cDNAs were amplified using QPCR. The amplification was performed using a degenerate forward primer corresponding to the highly 2+ conserved phosphate/Mg binding domain and a specific reverse primer; oligo(dA)18. A DNA fragment approximately 770 bp in length was obtained and cloned into a pTZ57R vector. This fragment was sequenced and the amino acid sequences were predicted from the nucleotide sequence. The protein sequence representing the -CC- motif (the geranylgeranylation region) at the Cterminal was found to be present in this Rab sequence. After the entire sequence was cloned and analyzed, this cDNA fragment was found to belong to the Rab7 family for japonica rice and was designed as OsRab7. The nucleotide and deduced protein sequences of OsRab7 are shown in Figure 1. This cDNA is 1,010 bp in length and contains 65 bp of 5’-untranslated region, an open reading frame of 628 bp, including 317 bp of a 3’untranslated region (excluding the poly-A tail). The ATG codon was located at position 66 and the TGA codon at position 691. The polypeptide of 206 amino acids had a calculated molecular mass of 23 kDa. Characterization Sequences of the Deduced Amino Acid A homology search was done using Blast which found that the deduced amino acid sequence of OsRab7 had strong homology to many small GTP-binding proteins (Rab7) in the Rab family from various plant species. The sequence was also aligned with ClustalW (Altschul et al., 1990) which showed that the sequence of Rab7 showed high homology to Rab7 from other plants [97% to Oryza sativa (japonica cultivar group), 90% to Cucumis sativus, 89% to Arabidopsis thaliana, and 88% to Lotus japonicus], with the highest score being to Rab7 from Oryza sativa (indica cultivar group) (98% identity). The multiple alignment result also revealed that the conserved region, including the Rab specific motif (RF), Rab subfamily regions (RSF), the guanine and 2+ phosphate/Mg binding site (G and PM) and double cysteine C-terminal motif were all contained in the OsRab7 sequence (Figure 2). Expression Profile Analysis of Osrab7 in Thai Jasmine Rice Tissues The expression of OsRab7 was examined using QPCR. The results revealed that OsRab7 was expressed in all rice tissues: roots, leaves, flowers and seeds [Figure. 3(A)]. The relative level of expression was analyzed from the intensity of bands using the Scion Image program [Figure. 3(B)]. From Figure. 3. the expression of OsRab7 was monitored in all rice tissues tested indicating that OsRab7 plays a role in several development processes in Thai jasmine rice. However, the results revealed an approximately 3 fold up-regulation of OsRab7 mRNA in 50 DAG leaves as opposed to senescing leaves than in 20 DAG leaves or young leaves. Thus, the expression of OsRab7 was examined in leaves treated with stressinducible senescence conditions to find out whether OsRab7 was involved in the senescence process in Thai jasmine rice. Expression Profile Analysis of Osrab7 Under Stress Induced-Senescence Conditions The Expression of OsRab7 was determined in Thai jasmine rice leaves under induced-stress conditions. The QPCR reactions revealed that the expression of OsRab7 was 2 - 6 fold up-regulated in the rice leaves exposed to 100 and 200 mM of NaCl for 3 and 5 days, 100 µM of ABA which showed the highest expression at 12 h, and kept in darkness for 3 and 5 days and for wounded leaves. Whereas, it was only slightly detected in leaves exposed to 1 mM of ethephon (Figure 4). 112 Int. Res. J. Plant Sci. 1 acctcGtcttcccgttccccgcgccgcgcgggctcgctccccgcgggggcagcttctaga 61 tcccgATGGCCTCCCGCCGCCGCACCCTACTCAAGGTCATCATCCTGGGCGACCCGGGGG M 121 A S R R R T L L K V I I L G D P G V TTGGGAAGACGTCCCTGATGAACCAATATGTGAACAAGAAGTTCAGCAACCAGTACAAGG G K T S L M N Q Y V N K K F S N Q Y K A 181 CTACGATTGGCGCGGATTTCCTCACCAAGGAGGTTCAGTTCGAGGATAGGCTCTTCACTT 241 TGCAAATATGGGATACTGCTGGCCAGGAAAGGTTTCAGAGTCTTGGTGTTGCATTCTACC T Q 301 I G W A D D T F A L G T Q K E E R V F Q Q F S E L D G R V L A F F T Y L R 79 A D C C V L V Y D V N S M K S F D N L 99 N W R E E F L I Q A S P S D P D N F P 119 CTTTTGTTCTTTTGGGCAACAAAGTTGATGTAGACAGTGGCAACAGCCGTGTGGTCTCTG F V L L G N K V D V D S G N S R V V S E 481 AGAAGAAGGCAAAGGCATGGTGTGCCTCTAAAGGGAATATCCCATACTTTGAGACATCTG 541 CCAAGGATGGTACAAACGTGGAGGAGGCTTTCCAGTGCATTGTAAAGAATGCTCTGAAGA K K 601 59 TTAACAACTGGCGTGAAGAATTTCTAATTCAGGCAAGCCCATCAGACCCTGATAACTTCC N 421 I 39 GTGGAGCAGATTGCTGTGTTCTAGTTTATGATGTCAATTCTATGAAGTCATTTGATAATC G 361 19 K D A G K T A N W V C E A E S A K F G Q N C I I P V Y K F N E A T L S K A N 139 159 179 ATGAACCAGAGGAAGAACTGTATGTGCCGGACACCGTGGATGTGGTGGGTGGCAACCGGC E P E E E L Y V P D T V D V V G G N R P 661 CCCCAAGATCATCCCGCTGCTGCTAGgacgtgatggaccatgaggggccagactgttggc 721 tatgcggtaacagaactacctttccacattgctgtgccaccatggtacctctcaaggacc 781 cattcgtaaccttttcaatcacctcatgtacccaattaagattgatgcgtctggcctgag 841 ttgtcaaatttgtggatgttgtgcaatttaggggtagcgtcatatctttgtgaatacaat 901 cggtgaaataagatgagtgtaaactgaagtttctccattatggttctctctgaaacgaac 961 aagatgaaattgttctgtctgcattgaggctgaaaaaaaaaaaaaaaaaa P R S S R C C Stop 199 206 Figure 1. The nucleotide sequence and deduced amino acid sequence of OsRab7. Bold letters indicate the start and stop codons, while the 5’ and 3’ UTR are indicated by lower case letters. The predicted amino acid sequence is shown below the nucleotide sequence in single-letter code. The CC- motif is underlined. The primer binding site and direction to amplify the partial cDNA is indicated by the arrows. Stress Induced-Senescence Conditions Reduced Chlorophyll Content in Thai Jasmine Rice Leaves The chlorophyll content in the leaf tissues was one indicator of the senescence parameter. Stress conditions such as NaCl, ABA, ethephon, darkess and wounding were chosen to induce a decrease in chlorophyll content of the rice leaves. The chopped leaf samples under stress conditions were then used to determine the chlorophyll content which is a parameter related to the senescence process. The chlorophyll content remaining in the leaf samples under stress conditions were then measured using the method and analysis first described by Porra (2002). The results showed that the chlorophyll content decreased in leaves (Figure 5) with the highest expression levels of OsRab7 as is shown in Figure 3. Expression Profile Analysis of Osrab7 in Thai Jasmine Rice Leaves Under Natural Senescence Conditions Since OsRab7 showed higher expression in senescing leaves than in young leaves (Figure 3.) and was Pitakrattananukool et al. 113 LoRab7 OsRab7 Os_inRab7 Os_jaRab7 CuRab7b AtRab71 LoRab7 OsRab7 Os_inRab7 Os_jaRab7 CuRab7b AtRab71 LoRab7 OsRab7 Os_inRab7 Os_jaRab7 CuRab7b AtRab71 LoRab7d OsRab7 Os_inRab7 Os_jaRab7 CuRab7b AtRab71 G1 RF1 PM1 RSF2 PM2 RF2 RSF1 MASRRRMLLKVIILGDSGVGKTSLMNQYVNRKFSNQYKATIGADFLTKEVQFEDRLFTLQ MASRRRTLLKVIILGDPGVGKTSLMNQYVNKKFSNQYKATIGADFLTKEVQFEDRLFTLQ MASRRRTLLKVIILGDTGVGKTSLMNQYVNKKFSNQYKATIGADFLTKEVQFEDRLFTLQ MASRRRTLLKVIILGDSGVGKTSLMNQYVNKKFSNQYKATIGADFLTKEVQFEDRLFTLQ MPSRRRTLLKVIILGDSGVGKTSLMNQYVNKKFSNQYKATIGADFLTKEVQFEDRLFTLQ MPSRRRTLLKVIILGDSGVGKTSLMNQYVNKKFSNQYKATIGADFLTKEVQFEDRLFTLQ *.:*** *********.*************:********************::******* PM3 RF3 RF4 IWDTAGQERFQSLGVAFYRGADCCVLVYDVNVMKSFDNLNHWREEFLIQASPSDPENFPF IWDTAGQERFQSLGVAFYRGADCCVLVYDVNSMKSFDNLNNWREEFLIQASPSDPDNFPF IWDTAGQERFQSLGVAFYRGADCCVLVYDVNSMKSFDNLNNWREEFLIQASPSDPDNFPF IWDTAGQERFQSLGVAFYRGADCCVLVYDVNSMKSFDNLNNWREEFLIQASPSDPDNFPF IWDTAGQERFQSLGVAFYRGADCCVLVYDVNSMKSFDNLNNWREEFLIQASPSDPENFPF IWDTAGQERFQSLGVAFYRGADCCVLVYDVNSMKSFENLNNWREEFLIQASPSDPENFPF ******************************* ***::**:******:*******:**** VVLGNKIDVDGGNSRVISEKKAKAWCASKGNIPYFETSAKEGFNVEAAFQCIAKNALKNE VLLGNKVDVDSGNSRVVSEKKAKAWCASKGNIPYFETSAKDGTNVEEAFQCIVKNALKNE VLLGNKVDVDSGNSRVVSEKKAKAWCASKGNIPYFETSAKDGTNVEEAFQCIVKNALKNE VLLGNKVDVDGGNSRVVSEKKAKAWCASKGNIPYFETSAKDGTNVEEAFQCIVKNALKNE VVLGNKVDVDGGNSRVVSEKKARAWCASKGNIPYFETSAKEGINVEEAFQCIAKNALKSG VLIGNKVDVDDGNSRVVSEKKAKAWCASKGNIPYFETSAKVGTNVEEAFQCIAKDALKSG *::***:***.****.:*****:***************** * *** **:**.::*:*. C PEEEMYLPDTIDVGGGGRQQRSTGCEC 207 PEEELYVPDTVDVVGGNRPPRSSRCC- 206 PEEELYVPDTVDVVGGNRAPRSSGCC- 206 PEEELYVPDTVDVVGGNRAQRSSGCC- 206 EEEEIYLPDTIDVGSNNQ-PRSSGCDC 206 EEEELYLPDTIDVGTSNQ-QRSTGCEC 206 *:::*:***:** .: * : * 60 60 60 60 60 60 120 120 120 120 120 120 180 180 180 180 180 180 Figure 2. Alignment of the deduced OsRab7 amino acid sequence from jasmine rice. Distinct functional domains are designed according to Pereira-Leal and Seabra (2001). Rab specific regions (RF), Rab subfamily specific regions (RSF), GDP/GTP-binding domains (G), Phosphate/M2+ binding domains (PM), and the geranylgeranylation region (C) are surrounded by boxes. The amino acid sequences were obtained from Oryza sativa [indica cultivar group] (Os_in), Oryza sativa [japonica cultivar group] (Os_ja), Cucumis sativus (Cu), Arabidopsis thaliana (At), and Lotus japonicas (Lo). Number refers to amino acid residues. Figure 3. The expression of OsRab7 in jasmine rice tissues. The expression of OsRab7 in various rice tissues; roots = R, the third leaf blades at 20 and 50 DAG = Ll and L4, flower = F, and seeds at 15 DAF = S (A). The relative expression level of OsRab7 in the rice tissues, presented as the ratio of expression of OsRab7/β-Actin (B). β-Actin was used as an internal control. 114 Int. Res. J. Plant Sci. Figure 4. The expression of OsRab7 under stress conditions. The expression of OsRab7 in rice leaves incubated in 3 mM MES buffer pH 5.8 supplemented with 100 and 200 mM NaCl under continuous light for 3 and 5 days (A); supplemented with 100 µM of ABA for 0, 3, 6, 12, and 24 h (B); supplemented with 1 mM ethephon for 0, 3, 6, 12, and 24 h (C); or in leaves were kept in darkness for 0, 3 and 5 days (D); and wounded leaves at 0, 3 and 5 days after wounding (E). The relative expression level of OsRab7 in those rice tissues, presented as the ratio of expression of OsRab7/β-Actin (F-J). β-Actin was used as an internal control. The meaning of letter code; D = day, h = hour. Pitakrattananukool et al. 115 Figure 5. Chlorophyll content of rice leaf samples incubated under stress induced senescence conditions. The chlorophyll content of the rice leaf samples incubated in 3 mM MES buffer pH 5.8 supplemented with 100 and 200 mM NaCl under continuous light for 0, 3 and 5 days (A); supplemented with 100 µM of ABA for 0, 3, 6, 12, and 24 h (B); supplemented with 1 mM Ethephon for 0, 3, 6, 12, and 24 h (C); or in leaves kept in darkness for 0, 3 and 5 days (D); and wounded leaves at 0, 3 and 5 days after wounding (E). The meaning of letter the code: D = day, h = hour. upregulated in most of the stress induced-senescence conditions except ethylene, its expression was determined in leaves under natural senescence conditions. The QPCR results revealed that the expression levels of OsRab7 showed the highest expression in leaf blades at 40 and 50 DAG [Figure 5(A)]. The relative level of expression was also analyzed from the bands intensities [Figure 5(B)]. This shows that the OsRab7 mRNA levels increased during leaf senescence periods. In this study OsRab7, was isolated from Thai jasmine rice and examined with an expression analysis. The result shows that the amino acids sequence retained the Rab specific domains (Figure 2). Interestingly, OsRab7 showed the highest expression levels approximately 3fold up-regulated in senescing leaves over young leaves and upregulated in leaves that were incubated in most stress induced-senescence conditions. Moreover, the decrease in chlorophyll content under the stress inducedsenescence was strong evidence that when leaves were senescing, the expression of OsRab7 was high. In addition, the OsRab7 sequence showed high similarity (97%) to OsRab7B3 isolated from japonica rice which is known to enhance leaf senescence in transgenic rice (Pitakrattananukool et al., 2012). Therefore, OsRab7 is a senescence-related gene likely to be involved in the chlorophyll degradation process during leaf senescence in Thai jasmine rice. For further analysis, OsRab7 gene 116 Int. Res. J. Plant Sci. Figure 6. The expression of OsRab7 in jasmine rice leaves under natural senescence conditions. The expression of OsRab7 in the third leaf blades at 20, 30, 40 and 50 DAG = Ll, L2, L3, and L4 respectively (A). The relative expression levels of OsRab7 in those rice tissues, presented as the ratio of expression of OsRab/β-Actin (B). βActin was used as an internal control. transfer is required to further understand its function in the senescence process. ACKNOWLEDGMENTS This work was granted by the Office of the Higher Education Commission, Thailand; Ms. Sugunya Pitakrattananukool was supported by a CHE PhD. Scholarship. REFERENCES Agarwal P, Reddy MK, Sopory SK, Agarwal PK (2009). Plant Rabs: characterization, functional diversity, and role in stress tolerance. Plant. Mol. 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