International Conference on Mathematics and Natural Sciences (ICMNS) 2008

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Proceedings

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PROCEEDINGS

of The 2

nd

International Conference on

Mathematics and Natural Sciences

(ICMNS) 2008

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The Second International Conference on Mathematics and Natural Sciences (ICMNS 2008) is organized by Faculty of Mathematics and Natural Sciences, School of Pharmacy, and School of Life Sciences and Technology at Institut Teknologi Bandung.

Organizing Committee

• Dr. Rinovia Mery Garnierita Simanjuntak (Chair)

• Dr. Ernawati Arifin

• Dr. Ayda Trisnawati Yusuf

• Dr. Bambang Prijamboedi

• Dr. Heni Rachmawati

• Dr. Hesti Retno Tri Wulandari

• Dr. M. Ikbal Arifyanto

• Dr. Siti Khodijah Chaerun

• Dr. Jalina Widjaja

• Dr. Sparisoma Viridi

Scientific Committee

• Prof. Bernward Bisping, University of Hamburg, Germany (Biotechnology)

• Prof. Shin Mineshige, Kyoto University, Japan

• Dr. Jai Prakash, University of Groningen, The Netherlands

• Dr. Yaya Rukayadi, Yonsei University, South Korea

• Prof. Dr. Edy Tri Baskoro, Bandung Institute of Technology, Indonesia

• Prof. Dr. Elin Yulinah, Bandung Institute of Technology, Indonesia

• Prof. Dr. Ismunandar, Bandung Institute of Technology, Indonesia

• Prof. Dr. Yeyet Cahyati Sumirtapura, Bandung Institute of Technology, Indonesia

• Dr. Ing. Cynthia L. Radiman, Bandung Institute of Technology, Indonesia

• Dr. Freddy Permana Zen, Bandung Institute of Technology, Indonesia

• Dr. I Nyoman P. Aryantha, Bandung Institute of Technology, Indonesia

• Dr. Mahasena Putra, Bandung Institute of Technology, Indonesia

• Dr. Pudji Astuti, Bandung Institute of Technology, Indonesia

• Dr. Sukrasno, Bandung Institute of Technology, Indonesia

• Dr. Suprijadi, Bandung Institute of Technology, Indonesia

• Dr. Taufiq Hidayat, Bandung Institute of Technology, Indonesia

• Dr. Tjandra Anggraeni, Bandung Institute of Technology, Indonesia

• Dr. Wardono Niloperbowo, Bandung Institute of Technology, Indonesia

Editor Committee

• Dr. Bambang Prijamboedi

• Dr. Rinovia Mery Garnierita Simanjuntak

• Dr. Ernawati Arifin

• Dr. Ayda Trisnawati Yusuf

• Dr. Heni Rachmawati

• Dr. Sparisoma Viridi

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This proceeding contains the papers of the talks presented in the Second International Conference on Mathematics and Natural Sciences (ICMNS 2008). The conference was jointly hosted by Faculty of Mathematics and Natural Sciences, School of Pharmacy, and School of Life Sciences and Technology at Institut Teknologi Bandung. This conference is also one of the many events organized to commemorate the 50th_{anniversary of Institut Teknologi Bandung.}

ICMNS 2008 was held in Aula Barat, Aula Timur, Campus Centre, and Basic Science Center at Institut Teknologi Bandung on 28-30 October 2008. The conference aims to promote interdisciplinary research in science and technology, to promote the development of science and their roles in the development of science-based technology, and to disseminate research in various field of mathematics and natural sciences.

There are nine main areas of research covered in this conference. They are health sciences, environmental sciences, biosciences and biotechnology, physical sciences, material sciences, mathematics, computer science and computational science, instrumentation, and earth and space sciences. Some of the papers were part of four mini-symposia: Biological Control, Computational Science, Graph Theory and Combinatorics, and Stellar Physics.

We would like to thank the invited speakers and those who have submitted papers to this conference. The organizer is grateful to Institut Teknologi Bandung for its support towards the organization of the conference. We would also like to thank Ikatan Alumni Institut Teknologi Bandung who had contributed for ICMNS 2008 presentation and poster awards.

Bandung, 14 June 2009

On behalf of the Organizing Committee, Rinovia Simanjuntak

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ISOLATION AND CHARATERIZATION OF

PHALAENOPSIS

ORCHID HOMEOBOX1(POH1)

cDNAS,

KNOTTED1-LIKE

HOMEOBOX FAMILY OF GENES

IN

PHALAENOPSIS AMABILIS

ORCHID

Endang Semiarti1_{, Takaaki Ishikawa}2_{, Yasushi Yoshioka}3_{, Masaya Ikezaki}3

Yasunori Machida

Abstract. The diversity of plant form derives from meristems, which are groups of indeterminate cells that produce organs from their flanks. The knotted1-like homeobox (knox) family of genes is one of the tool sets used in meristem function. To understand the shoot formation in Phalaenopsis amabilis (L.) Bl. which is one of Indonesian natural orchid, we isolated and characterized the cDNAs from its protocorms (developing orchid embryo). The synthesized cDNA(s) from six weeks old protocorms were cloned in Lambda ZAP II cloning system. Screening was carried out using 1.2 kb DNA fragment of KNAT1 gene from a model plant Arabidopsis. Finally, we got 10 putatitive cDNA clones with 400-500 bp in average lengths; designated as Phalaenopsis Orchid Homeobox1 (POH1) gene family. Using 5’- and 3’-Rapid Amplified cDNA Ends (RACE) system we got 1, 086 bp of the length of cDNA, that encodes 354 aa. The POH1cDNA sequences possess all of the features that characterize class 1 KNOX genes, including the highly conserved homeodomain with 100% homology to Dendrobium DOH1. Since Phalaenopsis and Dendrobium orchids have different pattern in shoot development, namely sympodial and monopodial, this data suggests that the homeodomain of POH1 and DOH1 function for shoot formation in very early stage of orchid development.

Keywords: KNOX, meristem, Arabidopsis, POH1, DOH1, Dendrobium, P. amabilis.

1 Introduction

In plant, shoot apical meristem (SAM) consists of a group of self-regenerating, indeterminate cells from which all above ground structures ultimately arise [1]. The developmental program is not restricted in any one phase of the life cycle, but rather new structures such as shoots, leaves, roots and flowers, they were produced continually. The angiosperm life cycle alternates between an extensive diploid phase and a most restricted haploid phase. The diploid phase is what we recognize as a plant. Its development consists of (i) embryogenesis; (ii) germination; (iii) primary development, in which shoots and roots elongate and branch; and (iv) secondary development, in which shoots and roots thicken, overlapping primary and secondary development [1]. The shift from vegetative to reproductive development is a significant transition in plant life cycle [2].

At present, almost every developmental process in the life cycle of plants is being scrutinised with molecular genetic tools. Homeobox genes are a universal group of developmentally important transcription factors that have been widely identified in animals, plants, fungi, and yeast [2]. The processes as complicated organ development can be described in operational terms as an ordered set of events directed by a few master regulatory genes [3]. In a model plant

Arabidopsis thaliana, KNAT1 gene was reported as a key gene for meristem-related gene

function in SAM [4]. Long et al. [4] detected the accumulation of the mRNA of KNAT1 gene is located in the SAM of Arabidopsis. Some leaf genes such as ASYMMETRIC LEAVES1, 2 (AS1, AS2) repress the expression of KNAT1 genes [4, 5]. In Arabidopsis, the switch ON and OFF of these two gene families regulates normal development of shoot system. Yu et al. [6] obtained

Dendrobium Orchid Homeobox1 (DOH1) gene from a hybrid plant of Dendrobium ‘Madame Thong

In’ as the only single heterologous gene of KNAT1 in dendrobium orchid.

The shoot system in orchid is categorized by two general forms of growth habit, i.e. monopodial and sympodial [7, 8]. Monopodial orchids grow upward from a terminal growth point and are tall, but not very wide, while sympodial orchids grow with some branching and produce leaves from the stems. Phalaenopsis orchids are the most common genus of cultivated monopodial orchids and P. amabilis has been used to create many Phalaenopsis hybrids that are grown as garden plants throughout the world, because of its desirable white, round flowers [7].

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Arabidopsis KNAT1 in the conserved region of homeodomain. Characterization of this gene will be discussed.

2 Methodology

Plant materials

Phalaenopsis amabilis (L.) Blume (Java form) was used in this study. The plant material was obtained from Royal Orchids (East Java, Indonesia). Seeds were sown on modified New Phalaenopsis (NP) medium (Islam et al., 1998). The cultures were maintained under continuous white light at 25˚C. The six -week-old protocorms were used for RNA isolation and cDNA synthesis.

Nucleic acid, cDNA synthesis and screening of cDNA library

Total RNA samples from vegetative shoot apical meristems (VSAMs; 6-week-old culture) were used for synthesis of cDNA(s). A cDNA library was constructed subsequently from the purified mRNA by using the Lambda ZAPII-cDNA Gigapack Cloning Kit (Stratagene, La Jolla, CA). The amplified cDNA library (>400,000 plaques) was screened under low-stringency conditions with the 32_{P labeling 1.2 kb-}_KNAT1_{cDNA probe by using a radioisotope labeling system (Amershams,}

USA) and detection kit (Boehringer Mannheim).

Sequencing and Sequence Analysis

Representative cDNA clones were sequenced on both strands by the dideoxynucleotide chain-termination method by using an ABI PRISM 377 DNA sequencer according to the manufacturer’s protocol (Perkin-Elmer, Foster City, CA). Alignment of deduced amino acid sequences was performed with the GENETYX-MAC Version 13.0.4 and CLUSTAL W program (Human Genome Center, Baylor College of Medicine, Houston, TX). Twenty two-amino acids of the homeodomains were used for phylogenetic analysis. Phylogenetic trees were constructed with the neighbor joining algorithm using the NEIGHBOR program.

3 Result and Discussion

Cloning and Sequence Analysis of a Class 1 KNOX Gene from Phalaenopsis amabilis Orchid

We constructed a cDNA library from developing orchid embryos (protocorms). The cDNA library derived from 6-week-old protocorms. The efficiency of cDNA library was high (87.5%), using PCR analysis we could amplified inserted cDNA in 14 out of 16 plaques (Figure 1).

Figure 1. High efficiency of P. amabilis orchid cDNA library. PCR analysis revealed about 87.5% plaques contained insert cDNA(s). (After Semiarti et al. [9])

Approximately 400,000 independent plaques were screened under low-stringency conditions. Using a 32_{P-labeled of 1.2 kb-full length KNAT1 cDNA we screened the orchid cDNA library. We}

obtained 22 positive cDNA clones in about 450-600 bp in lengths, that represent only one group of genes. Based on the conserved sequences in the homeodomain, we isolate the full length of POH1 cDNA using both 5’RACE and 3’RACE PCR. We got a 1086-bp cDNA clone, designated as

POH1 (Phalaenopsis Orchid Homeobox1). Comparison of POH1 deduced amino acids with a

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other angiosperm species, we constructed a phylogenetic tree based on analysis of the homeodomain region. Figure 3 shows that POH1 is placed in the same branch with DOH1, but is not grouped with its counterparts from any other angiosperm plants.

The high efficiency of cDNA library then was screened under low-stringency conditions for Class 1 KNOX homologous gene in orchid. Using a 32_{P- labeled of 1.2 kb-full lengths KNAT1 cDNA we}

screened the orchid cDNA library. We found the 22 positive cDNA clones with 410-500 bp in lengths. We search the homology of the isolated cDNA clones to other plants in sequence database and found that only 10 clones had homology to homeodomain in other plants including DOH1) that isolated by Yu et al. [6]. The clones, namely E2, E4, E6, E8, E16, E17, E18, E19, E21, E22 and E24 show homology to the evolutionary conserved region of homeodomain protein. Especially clone E6 had 100% similarity to DOH1 homeodomain. The other clones had similarity in range of 40-90% to other plant homeodomain. Therefore we classified the cDNAs into two Groups, Group I (E6) and Group II (E2, E4, E8, E16, E17, E18, E19, E21, E22 and E24). Based on the sequences of clone E6 that is a homologous of DOH1 cDNA, we performed 5’- and 3’-Rapid Amplified cDNA End (RACE) PCR. We got some clones, after cDNAs alignment, we got the longest rearranged cDNA clone, about 1,086 bp, designated as POH1 (Phalaenopsis Orchid Homeobox1).

Comparison of POH1 with a range of homeodomain proteins from other organisms such as Dendrobium and Arabidopsis revealed that POH1is a novel class 1 KNOX gene in P. amabilis

orchid, which is consists of 13 open reading frames (ORFs) with the well-conserved homeodomain (Fig. 2A). Figure 2B shows that POH1 homedomain protein is structurally 72% identical to DOH1 and had 74% similarity to KNAT1.

Figure 2. Structure and Evolutionary trees of POH1 gene. (A) Structure of POH1 gene with 3 frames that contains 13 ORF(s), encodes 354 a.a. (B-C) Evolutionary trees of POH1 gene with DOH1, KNAT1 and STM as gene counterparts, that shows 74% identity to DOH1 homeodomain, but separately grouping from KNAT1 and STM. E2-24 are the isolated clones of cDNAs. Only E6 clone has 100% sequence identity to DOH1. (After Semiarti et al. [9])

KNAT1 (Fig. 2B). As the characterization of across several plant species (for example,

Arabidopsis, rice or maize) can be used for isolation of orthologues and homologues from phylogenetically distant plants. The other nine cDNA clones, members of Group II show homology to KNAT1 cDNA (Fig. 3).

To elucidate the evolutionary relationship between the class 1 KNOX gene in orchid and those in other angiosperm species, a phylogenetic tree based on analysis of the homeodomain region.

A

B

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Figure 2C shows that the nine clones of isolated cDNA showed (20-90)% homology to each others, but outside grouped from both KNAT1 and STM.

Figure 3. Multiple alignment of KNOX domain of putative POH1 cDNAs, DOH1 and KNAT1. DOH1 -PDIASLLEEIRRENAGGERLAS-SSVI LGSDPELDEFMEMYCDVLVKYRRDLER--

KNAT1 PIQEAMEF I RR IE SQLSMLCQ- SP IHILNNPDGKSD--NMGSSDEEQENN-SGG 104

DOH1 GEAPESNLKGEERDLKEKLLRKYSGYLSSLKQEF--- 133 E6/POH1 LDAPESFLKGEERDLKEKLLRKYSGYLSSLKQEFKKK 135 E2 EAEVPEIDPRAEDKELKLHLLKKYSGYLS--- 142 E18 EAEVPEIDPRAEDKELKLHLLKKYSGYLSSLRQELSKKKK 136

E17 EAEVPEIDPRAEDKELKLHLLKKYSGYLSSLRQELSKKKK 147

E8 EAEVPEIDPRAEDKELKLHLLKKYSGYLSSLR--- 138

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293

The high similarity of amino acid sequences between DOH1 and POH1 cDNA, mainly in the KNOX1 domain is very interesting. Fig. 3 shows some amino acid sequences different in residue 1, 5, 26, 27 105 and 106 between these two genes this data suggests that the homeodomain of

POH1 and DOH1 function for shoot formation in very early stage of orchid development for critical decision that the shoot should grow sympodial or monopodial. But it still need to be explored through the detail characterization of the full length of POH1 cDNA. This work is in progress.

4 Conclusion

1. Two groups of cDNAs that consist of 10 clones of putative cDNA clones for Class 1 KNOX genes homologous were isolated from P. amabilis orchid, namely POH1 gene family.

2. Group I POH1 cDNA is a homologous cDNA of DOH1 cDNA from Dendrobium orchid.

3. Group II POH1 cDNA is homologous cDNA of KNAT1 cDNA from Arabidopsis thaliana.

Acknowledgment

We would like to thank to Ir. Sutikno Linuhung (Royal Orchids Nursery) for the gift of plant materials. E. S. was supported by a grant from the Ministry of Research and Technology of the Republic of Indonesia on the Project RUT IX (No. 14.15/SK/RUT/2004). This work was supported in part by Grants-in Aid for Scientific Research on Priority Areas (no. 14036216 to Y. M. and 16027250 to C. M.) from the Ministry of Education, Science, and Culture and Sports (MEXT) of Japan, by Core Research For Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation, and by “Academic Frontier” Project for Private Universities: matching fund subsidy from MEXT, 2005-2009.

References

[1]

S. Howell (1998), Molecular Genetics of Plant Development, Cambridge, UK, Cambridge University Press.

[2]

M. K. Ritter, C. M. Padilla, and R.J. Schmidt (2002), The Maize Mutant Bbarren stalk1 is Defective in Axillary Meristem Development. American Journal of Botany89(2): 2003-2010.

[3]

T. R. Burglin (1994), A Comprehensive Classification of homeobox In Guidebook to the Homeobox Genes, D. Double, ed., , Oxford Univ. Press.

[4]

M. Byrne, R. Barley, M. Curtis, J.M. Arroyo, M. Dunham, A. Hudson, and R. Martienssen (2000), Asymmetric leaves mediates leaf patterning and stem cell function in Arabidopsis,

Nature, 408, 967-971.

[5]

E. Semiarti, Y. Ueno, H. Tsukaya, H. Iwakawa, C. Machida, Y. Machida (2001) The

ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana regulates formation of a symmetric

lamina, establishment of venation and repression of meristem-related homeobox genes in leaves, Development 128, 1771-1783.

[6]

H. Yu, S.H. Yang, and C.J. Goh (2000). DOH1, a Class 1 knox Gene, Is Required for Maintenance of the Basic Plant Architecture and Floral Transition in Orchid, The Plant Cell 12, 2143-2159.

[7]

J. Arditi (1992), Fundamentals of Orchid Biology, John Wiley and Sons, New York.

[8]

M. Suryowinoto (1984), Mengenal anggrek-anggrek spesies, Fakultas Biologi Universitas Gadjah Mada.

[9]

E. Semiarti, T.Ishikawa, Y.Yoshioka, M.Ikezaki, Y. Machida and C.Machida (2008). Screening of cDNAs for KNOX1 homologous genes in Phalaenopsis amabilis orchid.

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ENDANG SEMIARTI

Laboratory of Plant Tissue Culture, Faculty of Biology,

Gadjah Mada University, Indonesia E-mail:

TAKAAKI ISHIKAWA

Plant Biology Research Center,

College of Bioscience and Biotechnology,

Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan E-mail:

YASUSHI YOSHIOKA

Division of Biological Science,

Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan E-mail:

MASAYA IKEZAKI