JUNE_2014.cdr Journal of Tropical Crop Science (ISSN 2356-0169; e-ISSN 2356-0177) is published four-monthly by Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University, INDONESIA. Publication details, including instructions for authors and subscription information: www.j-tropical-crops.com Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by the Publisher. The accuracy of the Content should be independently veri�ed with primary sources of information. The publisher shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. Permission to make digital or hard copies of part or all of a work published in Journal of Tropical Crop Science is granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage. ©Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University, INDONESIA. All rights reserved. Journal of Tropical Crop Science Volume Number 1 20141 June ON THE COVER The cover image shows sun�owers by Darda Effendi EDITORIAL BOARD Krisantini Sintho Wahyuning Ardie Sandra A. Aziz Robert J. Hampson Satriyas Ilyas Tri Koesoemaningtyas Rohana P Mahaliyanaarachchi Awang Maharijaya Maya Melati Roedhy Poerwanto Bambang Sapto Purwoko Sudarsono Muhamad Syukur Hugo Volkaert Malcolm Wegener Managing Editor Krisantini Graphic Design Syaiful Anwar Features Editor Damayanti Buchori Dadang Sisir Mitra Agus Purwito Ernan Rustiadi SHORT COMMUNICATION Tropical and Subtropical Fruits in India Sisir Mitra Heliconia Cultivar Registration Dave Skinner, Jan Hintze, Bryan Brunner RESEARCH ARTICLES Estimation of Genetic Parameter for Quantitative Characters of Pepper ( L.)Capsicum annuum Muhamad Syukur, Syaidatul Rosidah Irrigation Volume Based on Pan Evaporation and Their Effects on Water Use Ef�ciency and Yield of Hydroponically Grown Chilli Eko Sulistyono, Abe Eiko Juliana Evaluation of Commercial Sun�ower (Helianthus annuus ) Cultivars in Bogor, Indonesia, forL. Ornamental and Nursery Production Syarifah Iis Aisyah, Khotimah, Krisantini Different Growth Partitioning and Shoot Production of Talinum triangulare Treated with Organic and Inorganic Fertilizer Sandra Ari�n Aziz, Leo Mualim, Sitta Azmi Farchany Cloning and Characterization of P5CS1 and P5CS2 Genes from L. under DroughtSaccharum officinarum Stress Hayati Minarsih Iskandar, Dwiyantari Widyaningrum, Sony Suhandono Journal of Tropical Crop Science (ISSN 2356-0169; ISSN 2356-0177) is published four-monthly (one volume per year) bye- Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University, IPB Darmaga Campus, Bogor, Indonesia 16680. Send all inquiries regarding printed copies and display advertising to or to Secretary, Department ofinfo@j‐tropical‐crops.com Agronomy and Horticulture; telephone/fax 62-251-8629353. Permission to Reprint: Permission to make digital or hard copies of part or all of a work published in isJournal of Tropical Crop Science granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage and that copies bear the full citation and the following notice on the �rst page: “Copyright Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University”. For all other kinds of copying, request permission in writing from Head of School, Department of Agronomy and Horticulture of�ce, IPB Darmaga Campus, Bogor, Indonesia 16680. © Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University. All rights reserved. Printed in the Republic of Indonesia.IN S T T T U I B O G O R PERTA N IA N Abstract Increasing world sugar demand might be ful�lled with extensi�cati n which include the use ofland o dry area. Development of drought tolerance and high productivity sugarcane could be byvariety achieved plant genetic engineering. Under drought condition, proline will be accumulated and functioned as an osmoregulator in plant. Δ -5-1-pyrroline carboxylate synthase (P5CS) is one of the important enzyme ins proline biosynthesis. This enzyme is encoded by P5CS gene family. We cloned two homologous genesP5CS from sugarcane, (Accession Number :SoP5CS1 KF178299) and (Accession Number :SoP5CS2 KF178300), which encode 729 and 716 amino acid polypeptide . The between these genes wass identity two 74% based on nucleotide sequence . Thes SoP5CS1 gene had 98% with (Accessionidentity SbP5CS1 Number : GQ377719.1) and had 99%SoP5CS2 identity with (Accession Number : EF113257.1). In thisSaP5CS experiment, sugarcane were exposed toplantlets medium containing PEG 6000 (40%) for 12, 24, 48, and 72 hours. roline concentrationP was measured after treatment genes were zedand expression analy by real time qPCR. The results showed that the proline- concentration was increased 12 folds (9 8 umol g ) after. . -1 48-hours stress treatment. ighest expression ofThe h S o P 5 C S 1 o c c u r e d a t 2 4 - h o u r s t r e a t m e n t w i t h approximately 16 times from plant without PEG (control plant) and decreased gradually at 48 and 72 hours treatment. ighest expression of occuredThe h SoP5CS2 at 24-hours drought stress with approximately 3 6 folds. compared to control. In drought treatment, the expression level was higher than and hasSoP5CS1 SoP5CS2 increased signi�cantly at 12-hours treatment. tI is suggested that the gene contributes moreSoP5CS1 signi�cantly to the production of proline during drought stress than . , could potentialSoP5CS2 SoP5CS1Hence y be used as a marker to screen sugarcane variety for drought tolerance for the development of transgenicand plant tolerant to drought. Keywords: cloning, drought, expression, P5CS, sugarcane Introduction Sugarcane is mainly used as raw material for sugar and bioethanol production. This crop productivity and growth has been in�uenced by enviromental factors such as climate and soil condition. Drought and salinity could decrease sugarcane productivity and growth (Silva et al., 2011). In contrast, the use of marginal and barren land for crop plantation is needed to ful�ll high world sugar demand. Research to improve plant stress tolerance is crucial to boost the sugarcane productivity and extend its range. Plant genetic engineering is one of the potential methods for improving plant quality. Proline accumulation is an adaptive response when plants are exposed to abiotic stresses. Proline acts as osmotic adjustment and osmoprotectant which function to maintain homeosta condition, stabilize cellsis structure, and prevent protein degradation (Verbruggen and Hermans, 2006; Taiz and Zeiger, 2006). Glutamate (Glu) and ornithine (Orn) are the proline precursors (Verbruggen and Hermans, 2006; Roosens et al., 1998). Glutamate pathway is the main route for proline biosynthesis during abiotic stresses. In glutamate pathway, proline is synthesized from glutamate via -ɣ glutamic semialdehyde (GSA) and Δ -pyrroline-5-1 carboxylate. This process is catalyzed by two enzymes, Δ -pyrroline-5-carboxylate synthethase (P5C ) and Δ -1 1S pyrroline-5-carboxylate reductase (P5CR) (Verbruggen and Hermans, 2008; Hare and Cress, 1997; Kishor et al., 2005). P5CS, a rate-limiting enzyme, plays an important role in proline biosynthesis. P5CS is a bifunctional enzyme that exhibits -glutamyl kinase and glutamic- -semialdehydeɣ ɣ dehydrogenase activities. This enzyme is encoded by two homologous genes (Verbruggen andP5CS Hermans, 2008; Hare and Cress, 1997; Kishor et al., 2005). The genes had been cloned andP5CS characterized from several plants such as Arabidopsis thaliana , Solanum licopersicum(Savoüre et al., 1995) (Fujita et al., 1998) ( et al., ;, Oryza sativa Igarashi 1997 Hur 2004 Zhu 1998et al., ; et al., ) and, Sorghum bicolor ( et al ). In some species, these genes showedSu ., 2011 different expression patterns. Both of genes hadP5CS different functions in plant development stage and stress response ( and ).Verbruggen Hermans, 2008 In , the expression of was induced byA thaliana AtP5CS1. drought and salt stress while was ubiquitouslyAtP5CS2 expressed in dividing and differentiating cells ( etSavoure al., ). had two closely related1995 S licopersicum P5CS. RESEARCH ARTICLE Cloning and Characterization of and Genes fromP5CS1 P5CS2 Saccharum officinarum L. under Drought Stress Hayati Minarsih Iskandar Dwiyantari Widyaningrum Sony Suhandono AC B , , B ABiotechnology Research for Estate Crop, Jalan Taman Kencana No. 1, Bogor, IndonesiaInstitute Genetics and Molecular Biology Division, School of Life Science and Technology, Institut Teknologi Bandung,B Jalan Ganesha No. 10, Bandung, Indonesia C Corresponding author; email: hmiskan@yahoo.com 5555 23 Journal of Tropical Crop Science Vol. 1 No. 1, 2014June www.j-tropical-crops.com genes, and . The expression oftomPRO1 tomPRO2 tomPRO1 was induced by salt stress the, whilst tomPRO2 transcript was undetec able (Fujita et al.,t 1998). In , gene was a housekeepingO sativa OsP5CS1. gene while OsP5CS2 was induced by salt, drought, and, cold stress ( et al., ; et al., ; etIgarashi 1997 Hur 2004 Zhu al., ). In , the transcript was1998 S bicolor SbP5CS1. signi�cantly up-regulated under abiotic stress while the SbP5CS2 was a housekeeping gene which function in proline biosynthesis under normal condition (Su et al., 2011). Previous study showed that overexpression of VaP5CSfrom Vigna aconitifolia increased stress tolerance and proline content on transgenic tobacco ( et al., ).Kishor 1995 This study aims to characterize twoclone and homologous P5CS genes and �nd the more potential canditate gene that might be used to improve drought tolerance in sugarcane. In this research, two homologous P5CS genes had been cloned from sugarcane followed by its characterization using bioinformatic analysis. We studied the expression pattern of genes duringSoP5CS drought stress and demonstrated its relation with proline accumulation to �nd the more potential candidate among those genes for sugarcane genetic engineering. Material and Method Plants materials and drought stress treatment For cloning full-length isolat on of andand i SoP5CS1 SoP5CS2 genes, month old�ve after acclimatization of sugarcanes were not wateredplants of PSJT 941 variety in seven days in order to simulate drought conditions. The sugarcanes were planted in polybags containing a mixture of , sand and manure The control plants weresoil . watered until sampled T were used fhree-month old sugarcane planlets or expression study of and genesSoP5CS1 SoP5CS2 under drought stress and proline measurement. The planlets were incubated in [MS+PEG] medium in 12, 24, 48, and 72 hours in order to simulate drought stress. We used PEG 6000 with 40% (w/v) concentration. For control, the planlets were incubated in MS medium in 12, 24, 48, and 72 hours. The leaf samples were collected after incubation three biological replicates for eachwith treatment. Isolation of total RNA and cDNA synthesis Total RNA was extracted from leaf samples using Total R N A ( p l a n t ) I s o l a t i o n K i t G e n e a i d f o l l o w i n g manufacturer's intruction. cDNA was synthesized using Revert-Aid cDNA synthesis Kit Fermentas and Super( ) Script III cDNA Synthesis Kit Invitrogen according to the manufacturer's instruction. Cloning of SoP5CS1 and SoP5CS2 genes We used two different primer pairs (P5CS1-F: 5'- G A G C C A C T TA G C G A G G A A G - 3 ' ; P 5 C S 1 - R : 5 ' - G G T C C T T C T G C T T G T TA C G A - 3 ' ; P 5 C S 2 - F : 5 ' - AGAGGGGAGACCGAA GACCAGGAG-3'; P5CS2-R: 5'-TACAGAATGAACCACCAGAATGAT-3') in order to clone the SoP5CS1 and SoP5CS2 genes from sugarcane. Primers were designed using Primer3 (www.frodo.wit.mit.edu). Primer P5CS1-F and P5CS1-R were designed based on EST database number T20007 : 9719 from KEGG (Kyoto Encyclopedia of Genes and Genomes) and nucleotide sequence of geneSbP5CS1 (Genbank Accession Number : GQ377719.1). Primer P5CS2-F and P5CS2-R were designed based on SoP5CS (Genbank Accession Number : EF155655.1). The 50 uL PCR mixture contained 2 uL (250 ng) cDNA, 0 2 mM dNTP, 0 5 uM primers, 5 uL 10X. . KAPA2G GC Buffer ( 1.5 MgCl KAPA2Gcontained mM ), and 1 unit2 Robust Hotstart DNA Polymerase. We used touchdown PCR program that was set as followed: initial denaturation at 95 C for 5 minutes, 10 cycles of 95 C foro o 30 s, 60 C for 30 s, 72 C for 3 minutes, 10 cycles ofo o touchdown program (95 C in 30 s, annealing temperatureo between 60 C to 50 C in 30 s, 72 in 3 minutes), 10 cycleso o for 95 C for 30 s, 50 C for 30 s, 72 C for 3 minutes,o o o folllowed by �nal extension of 72 C for 7 minutes. PCRo were done using Veriti Thermal Cycler Applied Biosystem. The fragment of gene was clonedSoP5CS1 to pGEM-T easy vector (Promega) while fragment of SoP5CS2 gene was cloned to pJET 1.2/blunt vector (Fermentas) then transformed to DH5 competentE.coli cells. The positive recombinant plasmids were subjected for sequencing. Bioinformatics We used several bioinformatic analysis methods in order to analyze the sequences and predict the P5CS proteins. Homology analysis of nucleotide sequence wasSoP5CS done using BLASTn. Multiple sequence alignment was made by ClustalW. Phylogenetic trees were made using clustalX and MEGA5 using Neighbor- oining method.J Amino acid sequence of protein weres SoP5CS predicted using Bioedit. In order to predict SoP5CS proteins celular localization, we used TargetP and ProtComp from Softberry (www.softberry.com). Real time q PCR (RT- PCR) analysisuantitative q RT- PCR was used for expression analysis. The 20 uLq RT- PCR mixture contained 10 uL 2X Power Sybr Greenq (Applied Biosystem), 0 1 uM primers, and 2 uL cDNA. template. We used different pairs of primer for each gene. F o r G A P D H g e n e , w e u s e d G A P D H - F ( 5 ' - TGCACCCATGTT CGTTGT-3') and GAPDH-R : 5'- CCATCAACAGTCTTCTGGGT-3'. -spesi�cSoP5CS primers were used as followed : (P5CSA-F: 5'- GAGCCACTTAGCGAGGAAG-3' ; P5CSA-R: 5'- TTCTGCCCAGTG ACAACAG-3') for andSoP5CS1 (P5CSB-F: 5'-GGAGACCGAAGACCAGGA-3' and P5CSB-R: 5'-TCACAATGATCACCTCGTACC-3') for SoP5CS2. We used relative quantitative Real Time PCR 5555 H , D Sayati Minarsih Iskandar wiyantari Widyaningrum, ony Suhandono24 Journal of Tropical Crop Science Vol. 1 No. 1, 2014June www.j-tropical-crops.com program that was set as followed : initial denaturation at 95 C in 10 minutes, 40 cycle of RT- PCR (95 C in 15s ando oq 57 C in 15s), followed by melt curve program witho different melting temperature (ΔTm = 0 3 C) at every. o cycle from 60 C to 90 C. Real time qPCR were doneo o using Applied Biosystem Step One Plus Real Time PCR. In RT-qPCR, we did three technical replicates for each genes. The relative expression ratio of andSoP5CS1 SoP5CS2 gene were calculated with pfaf� method (Pfaf�, 2001). The Pfaf� method was used because of the ef�ciency of the GAPDH primers as gene reference, was different with the ef�ciency of P5CS1 and P5CS2 primers. The ef�ciency of GAPDH, P5CS1, and P5CS2 primers were 1.8, 2.11, and 2.0. Proline determination Free proline was extracted and calculated using Bates (1973) method 0 5 gram fresh lea was homogenized: . ves in 10 mL of 3% sulfosalicylic acid. The homogenate was sentrifuged to separate supernatant (�ltrate) and solid molecul. 2 mL of �ltate was reacted with 2 mL acid- ninhidrin and 2 m glacial acetic acid. The mixtures wereL boiled in 100 for 1 hour, kept on ice, and then wereoC extracted using 4 mL toluene. Proline absorbance was measured using Life Science Spectrophotometer at λ 520 nm. Proline concentration was calculated using equation (i) : y = O ; proline concentration M). The equationD520 (μ was based on proline standard curve with R = 0.9987.2 We continued to measure proline concentration using equation (ii) based on Bates (1973) : Results Sequence Analysis and Phylogenetic Tree Two sugarcane genes were isolated from 5' UTR toP5CS 3' UTR region by RT-PCR and registered in NCBI (Genebank Accession Number : KF178299 and Genebank Accession Number : KF178300). The SoP5CS1 SoP5CS2and encoded 716 and 729 amino acid polypeptides which was predicted would be located in cytoplasm. Sequence analysis showed that SoP5CS1 shared 75 6% in nucleotide sequence and. identity s shared 75 7% similarity protein sequence with. s SoP5CS2 SoP5CS1. BLASTn results showed that shared 96% similarity with (GenebankSbP5CS1 Accession Number : GQ377719.1; protein id : ACU65226) while had 98% withSoP5CS2 identity SoP5CS (Genebank Accession Number : EF155655), 98% with (Genebank Accessionidentity SaP5CS Number : EU113257), and 93% withidentity SbP5CS2 (Genebank Accession Number : GQ377720). The two protein sequences were aligned withSoP5CS AtP5CS, OsP5CS, and SbP5CS. Multiple sequence alignment result (Figure 1) showed that both of SoP5CS protein sequences had conserved regions such as ATP binding site, glutamate-5-kinase (G5K) domain, NAD(P)H binding site, putative leucine zipper domain, and glutamate semialdehyde dehydrogenase (GSA-DH) domain. onserved Phe (F) residue also found inThe c SoP5CS1 SoP5CS2(position 141) and (position 128) protein sequence. The residue was functioned in proline feedback inhibition regulation ( et al ).Hong ., 2000 Phylogenetic tree (Figure 2) was made based on the P5CS protein sequences in order to study the evolutionary relationship. It was constructed by ClustalX and MEGA5 using NJ method with 1000 bootstraps. It shows that there were two clusters separating monocots and dicots P5CS protein. Two proteinsof areSoP5CS located within the monocot cluster owever these. H , proteins were in different sub-cluster which ds divide monocot P5CS1 and P5CS2. The sharedSoP5CS1 homology with SbP5CS1, ZmP5CS, and OsP5CS2 while SoP5CS2 similar to SoP5CS, SaP5CS, and SbP5CS2.is Expression Analysis Real time quantitative PCR (RT-qPCR) was performed in order to analyze the expression of andSoP5CS1 SoP5CS2 genes during drought stress. The expression of genes on drought treatment plants wereSoP5CS compared to control plant. Both genes were induced by drought stress. The expression of wereSoP5CS1 increased signi�cantly up to 11 folds at 12-hours drought treatment then continously increased to achive the highest ratio, 16 folds, at 24-hours treatment. However, SoP5CS1 transcript gradually decreased to 13 folds and 10 folds at 48-hours and 72 hours drought treatment (Figure 3a.). The expression of not signi�cantly increaseSoP5CS2 did during drought stress. transcript was inducedSoP5CS2 to achieve the highest ratio, 3.3 folds, at 24-hours drought stress. The transcript was down-expression of SoP5CS2 regulated to 1.4 folds at 48-hours drought stress. The gene expression was increased again up to 2.8 folds at 72-hours treatment (Figure 3b.). The expression of SoP5CS1 SoP5CS2was compared to treat plantof ed during drought condition (Figure 4). heHence, t expression of was signi�cantly higher thanSoP5CS1 SoP5CS2 SoP5CS. The expression pattern of genes were similar to genes.SbP5CS 55 25Cloning and Characterization of and Genes fromP5CS1 P5CS2 .......... Journal of Tropical Crop Science Vol. 1 No. 1, 2014June www.j-tropical-crops.com Figure 1. Multiple sequence alignment between two SoP5CS protein sequence with SoP5CS (protein id : ABM30223), SbP5CS1 (protein id : ACU65226), SbP5CS2 (protein id : ACU65227), OsP5CS1 (protein id : BAA19916), OsP5CS2 (protein id : BAG94966), AtP5CS1 (protein id : NP_001198715.1), AtP5CS2 (protein id : NP_191120.2). Boxes ([]) show predicted conserved region. 5555 H , D Sayati Minarsih Iskandar wiyantari Widyaningrum, ony Suhandono26 Journal of Tropical Crop Science Vol. 1 No. 1, 2014June www.j-tropical-crops.com Figure 2. Phylogenetic relationship of SoP5CS1 and SoP5CS2 (boxes) with saP5CS (S. arundinaceum P5CS), soP5CS ( P5CS), sbP5CS2 ( P5CS1), osP5CS1 ( P5CS1), taP5CSS. officinarum S. bicolor O. sativa ( P5CS), OsP5CS2 ( P5CS2), zmP5CS ( P5CS), sbP5CS ( P5CS1),T. aestivum O. sativa Z. mays S. bicolor vaP5CS ( P5CS), gmP5CS ( P5CS), jcP5CS ( P5CS), rcP5CS (V. aconitifolia G. max J. curcas R. communis P5CS), atP5CS1 ( P5CS1), and atP5CS2 ( P5CS2). The phylogenetic tree had twoA. thaliana A. thaliana cluster separating monocots (I) and dicots P5CS (II). A B Figure 3. The Expression of ( ) and ( ) in treatment and control plantSoP5CS1 SoP5CS2A B Figure 4. Comparison of two genes expression during drought treatmentSoP5CS 55 27Cloning and Characterization of and Genes fromP5CS1 P5CS2 .......... Journal of Tropical Crop Science Vol. 1 No. 1, 2014June www.j-tropical-crops.com Proline Concentration The proline concentration was measured using the same samples for expression analysis in order to determine the corelation between proline accumulation with SoP5CS genes transcript level. The proline content of treatment plant was higher than control plant since 12-hours drought treatment (Figure 5). The proline concentration was signi�cantly increased at 48-hours treatment then achieved the highest peak (9 8 umol gram ) at 72-hours. . -1 drought stress. Figure 5. Comparison of proline content between treatment and control plant Discussion The P5CS enzyme from sugarcane was encoded by two homologous genes as observed in other plantP5CS species Initial. , we cloned the genes that wereSoP5CS2 related with and genes databaseSoP5CS SbP5CS2 on in NCBI Genebank. We found another cDNA fromP5CS sugarcane using KGENES Search by KEGG (Kyoto Encyclopedia of Genes and Genome). This data shared high homology with gene from sweet sorghum.SbP5CS1 Both of the isolated genes had complete codingSoP5CS region which encode two closely related SoP5CSs protein .s The SoP5CS proteins shared high similarity with SbP5CS2 as shown in multiple alignment result. The SoP5CS1 SbP5CS1 SoP5CS2similar to whil had high, st homology with .These proteins had conservedSbP5CS2 motif ATP and NAD(P)H binding site, Glu-5-kinase and gamma-glutamyl phosphatase reductase domain, and leucine zipper region that were located at the same position (Su et al., 2011). The result indicated that the SoP5CS proteins would have same expressionthe pro�le with proteins.SbP5CS In phylogenetic tree, the P5CS protein from many plant species were clustered into two groups P5CSdividing on monocots and dicots. The andSoP5CS1 SoP5CS2 protein were clustered in different sub-groups within monocots P5CS. This result indicated that sugarcane P5CS protein was encoded by P5CS gene family. Turchetto-Zolet ( ) studied the evolutionary2009 relationship of P5CS protein. In their phylogenetic tree, monocots and dicots P5CS were clustered separately. Further study showed that there were two sub-clusters in monocots P5CS separating P5CS1 and P5CS2 protein. This event could result in one or more copies of a gene in genome which had new function or paralogous gene (Grassi et al., 2008). There were some previous studies about geneP5CS from , , and reportedA thaliana O sativa S bicolor. . . that about s ousexpression pro�le of two homolog P5CS genes from the plant species under abiotic stresses such as drought, salt, and cold. e focus on geneHere, w P5CS expression under drought stress. In ,A thaliana AtP5CS1. had a role in abiotic stress response and prolin accumulation. This gene expression increased during drought stress. AtP5CS2 was especially expressed in dividing cell. The gene was ubiquitously expressed both in normal and stress condition. However the expression of AtP5CS2 was signi�cantly lower than during abioticAtP5CS1 stress such as drought. In , the wasO sativa OsP5CS1. highly expressed in reproductive and vegetative organs especially during normal condition. In contrast, the OsP5CS2 transcript was highly induced by drought stress. The genes had similar expression pattern withSbP5CS AtP5CS OsP5CS SbP5CS1and genes. The gene was highly expressed in mature plant during abiotic stress including drought condition. The transcript wasSbP5CS2 highly detected in dividing cell. The was alsoSbP5CS ubiquitously expressed both in normal and drought condition however the expression of wasSbP5CS1 extremely higher than . Su ., (2011)SbP5CS2 et al suggested that had an important role in prolineSbP5CS1 5555 H , D Sayati Minarsih Iskandar wiyantari Widyaningrum, ony Suhandono28 Journal of Tropical Crop Science Vol. 1 No. 1, 2014June www.j-tropical-crops.com 55 29Cloning and Characterization of and Genes fromP5CS1 P5CS2 .......... accumulation during abiotic stress while SbP5CS2 played a role in proline biosynthesis duringregular normal condition. Transcriptionalor unstressed regulation is a major key in gene expression. Study about SbP5CS SbP5CSpromoters showed that both of genes had cis-elements such as MYCCONSENSUSAT, WRKY, and MYBCORE. These elements functioned as binding site for transcription factors which nvolved in cold,i wound, and drought stress ( et al., ). Two closelySu 2011 related P5CS genes had different function inS bicolor. plant development, growth, and stress response that might be caused the different regulation of -elementby cis in SbP5CS promoter ( et al , ).Alberts . 2008 Current study reported one type gene fromP5CS sugarcane (Iskandar et al 201 ; Patade et al., 2012).., 1 Iskandar et al (201 ) reported the expression of in. P5CS1 young and mature culm internodes of sugarcane after 15- days water de�cit treatment. The expression of P5CS was higher than control, and generally not signi�cantly different between the young and mature culm internodes. Expression analysis showed that both of andSoP5CS1 SoP5CS2 were induced by drought stress. The SoP5CS1 transcript was signi�cantly increased, when compared to control, during PEG incubat . However,ion the expression of was not signi�cantlySoP5CS2 increased during drought condition. Our study showed that transcript and proline accumulation hadSoP5CS1 different pattern. The expression of had beenSoP5CS1 signi�cantly increased at 12-hours drought treatment then achieved the highest peak at 24-hours drought treatment. transcript level was graduallySoP5CS1 decreased after 24-hours treatment. In contrast, proline was signi�cantly accumulated at 48-hours drought treatment.The highest proline concentration was achieved at 72-hours drought treatment. The expression and activity of P5CS protein might be inhibited by feed- back inhibition regulation of high proline concentration. The proline was continously produced after 48-hours, when transcript repressed, might be causedSoP5CS1 by proline biosynthesis by ornithine pathway. Study in A. thaliana showed that ornithine pathways played a role in proline biosynthesis during drought and salt stress ( et al. 1998).Roosens , Based on the similarity of SoP5CS with SbP5CS genes and expression analysis data, we suggest that the SoP5CS1 gene contributes more signi�cantly to the production of proline during drought stress than SoP5CS2 SoP5CS1. The could potential be used as aly marker to screen sugarcane variety for drought tolerance and may also potentially be useful for the development of tran genic plants tolerant to drought. Further study abouts SoP5CS promoters is needed in order to determine the SoP5CS1 SoP5CS2and gene regulation. Acknowledgement This research was supported by Research Colaboration between Indonesian Biotechnology Research Institute for Estate Crops ( )BPBPI , PT. Riset Perkebunan Nusantara (RPN), Indonesian Center for Estateand Crops Research and Development (PUSLITBANGBUN) with funding from DIPA APBN 2012. The Authors are also grateful to . 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