MOLECULAR IDENTIFICATION OF CSLA GENE ENCODING GLYCOSIL TRANSFERASE RESPONSIBLE FOR THE SYNTHESIS OF GLUCOMANNAN IN PORANG (Amorphophallus muelleri

Blume)
Estri Laras Arumingtyas1)
1)

Biology Department, Faculty of science, University of Brawijaya, Malang, Indonesia (larasbio@gmail.com)

Abstract
Porang (Amorphophallus muelleri Blume) is a member of the family Araceae which has a high glucomannan content. Glucomannan is used in the food industry (food healthy diet), paper industry, pharmaceuticals and cosmetics, and exported to Japan and China. Glucomannan synthesis in a variety of plants was regulated by many genes. One of the most important genes is glucomannan synthase gene of cellulose synthase-like A (CSLA) glycosyltransferase. From a previous study, it has been identified two lines of porang contained represented high and low glucomannan contents. However it has not been investigated whether the difference was caused by the existence or the difference of the sequence of CSLA gene. So, in this research, identification of CSLA gene was conducted using Polymerase Chain Reaction (PCR) technique, to detect the effect of CSLA gene to the glucomannan content of porang. The result showed that the glucomannan content of porang collected from Sumberbendo and Klangon were higher than suweg. Even Sumberbendo variant showed far more higher glucomannan content than Klangon. Whilst one of the two variants of suweg (Suweg 2, from Wlingi) showed higher content of glucomannan compared to the other variant. The difference of glucomannan content showed by variants of porang from different places could be caused by genetical and environtmental factors. The size of genomic DNA of porang which were different proved that there were genome variations found in porang(Amorphophallus mueleri Blume). CSLA gene was shown to be exist in porang and suweg. However the gene was only found in the the flower spatha and bulb, but was not found in the leaf of porang. The gene detected in suweg was different in number and size compared to porang. This difference maybe the cause of difference in the glucomannan content. Keywords: Porang (Amorphophallus muelleri Blume), CSLA gene, glycosil transferase, glucomannan.

I. INTRODUCTION Porang (Amorphophallus muelleri blume) is a member of the family araceae which has high glucomannan content [1]. Glucomannan is used in the food industry (food healthy diet), paper industry, pharmaceuticals and cosmetics. Production of porang corm for it is glucomannan content mostly to fulfill market demand of Japan and China. Glucomannan is a polysaccharide consisting hydrocoloid D-glucose and D-mannose. Glucomannan synthesis in a variety of plants was found to be regulated by many genes. Formation of guanosine 5'-diphosphate glycosil (GDP)-Dmannose requiring enzyme GDP-D-mannose pyrophosphorylase [2]-[4], while the formation of GDP-D--glucose requiring enzymes GDP-D-glucose pyrophosphorylase [5]-[6]. It was also reported the existence of an enzyme GDP-D-mannose 2epimerase that catalyzes the interconversion of GDP-D-mannose to GDP-D-glucose and vice versa. Subsequent formation of glucomannan chains made with the help of enzymes syntase glucomannan [7]. The members of glucomannan synthase enzymes that have been shown to play a role in the

formation of glucomannan in various parts of the plants is grouped into the family of cellulose synthase-like A (CSLA) glycosyltransferase. Research of genes that play a role in the formation of these enzymes in porang has not been reported. Information of the identity, structure and sequence of these genes will greatly assist identification of high yielding varieties in porang breeding. From previous study, it has been identified two lines of porang represented high and low glucomannan contents. However it has not been investigated whether the difference was caused by the existence or the difference of the sequence of CSLA gene. So, in this research, identification of CSLA gene was conducted using Polymerase Chain Reaction (PCR) technique, to detect the effect of CSLA gene to the glucomannan content of porang II. MATERIAL AND METHODS A. Plant Material and DNA Isolation Plant material used in this research were bulb, and flower spata of porang collected from Klangon which represent low glucomannan content, corm of porang collected from Sumberbendo represent high glucomannan content, and suweg collected from

Wlingi represent very low/zero glucomannan content. DNA genome was isolated from the corm, leaf, bulb and flower spatha of porang collected from Klangon , leaf of porang collected from Sumberbendo and corm of suweg from Wlingi. The methods used for isolation of genome DNA was Doyle and Doyle [8]. B. Polymerase Chain Reaction Polymerase Chain Reaction (PCR) was performed using a pair of specific primer designed based on the sequence of CSLA gene of Physcomitrella patens subsp. Patens. The sequence of the primers were forward- 3-ATGGCGGAGA GCGGGAGCAC-5 and reverse 3TCCACCCGCACAGACTGGCG-5. The PCR reaction mix consist of 2.5 l 10x buffer Ex Taq Polymerase, 2 l 25 mM MgCl2, 2 ul 10 mM dNTP, 25 pmol primer, 25 pmol Ex Taq Polymerase, and water to reach 25 l. The PCR program used were 93 C for 1 minute pre heating continued with 40 cycles consist of 93 C for 1 minute denaturation, 53 C for 1 minute annealing, and 72 C for 1,5 minute extension and finally ended 72 C for 10 minute final extension and 4 C for 5 minutes cooling. III. RESULT AND DISCUSSION A. Glucomannan content Measurement of glucomannan content (Table 1) showed that porang porang varian collected from Sumberbendo possess a much more high glucomannan content compared to the one from Klangon. Whereas suweg showed the lowest glucomannan content, except the one collected from Wlingi (Suweg 2). Table 1. Glucomannan content of porang dan suweg used in the experiment No. Varian Glucomannan content (% dry weight) 1. Sumberbendo 1 97.2 2. Sumberbendo 2 40.3 3. Sumberbendo 3 81.5 4. Klangon 1 44.7 5. Klangon 2 24.6 6. Suweg 1 16.2 7. Suweg 2 38.8 Porang or suweg that came from different site in the same village also showed different value of glucomannan content. There are two possibilities that cause that phenomenon to happen. The first possibility was genetic factor which also consist of two possibilities. The first possibility of genetic factor was all the porang anf suweg variants possess different sequence of CSLA genes which in turn will produce different kind of enzyme or enzyme

activity. Differences of the kind of enzyme or enzyme activity will produce different level of glucomannan content. This suggestion need to be proven by sequencing the CSLA genes of each sample. The second possibility of genetic factor is the enzyme was available which mean the gene was available and active, however as has been investigated before [9] the level of glucomannan resulted depend on the concentration ratio of GDPD-glucose and GDP-D-mannose available. There are many genes responsible for the formation of glucomannan. The components of glucomannan are mannose and glucose. The formation of glycosil guanosine 5'diphosphate(GDP)-D-mannose need the existence of GDP-D-mannose pyrophosphorylase enzyme [2][4], whereas the formation of GDP-D glucose need the presence of GDP-D-glucose pyrophosphorylase enzyme [5]-[6]. It was also reported GDP-Dmannose 2-epimerase catalyzed GDP-D-mannose to GDPD- glucose interconversion (Elbein 1969). Subsequent formation of glucomannan chains made with the help of enzymes glucomannan syntase [7]. The members of glucomannan synthase enzymes group that has been proven to have a role in the formation of glucomannan in many part of plants was the family of cellulose synthase-like A (CSLA) glycosyltransferase. There are nine members of CSLA gene family of Arabidopsis, and all of them were differentially expressed in various part of plants [10]. It was also showed that glycocil transferase is an active centre which capable of utilizing GDP-D-mannose or GDP-D-glucose to be synthesized to heteropolisaceride [10]. So, if there is a change in the sequence of CSLA gene, glucomannan chain can not be produced. The second possibility is the environmental factor. Environment where porang is growing affects porang content [11]. So even though porang have the same genes, but because it grows in a different environment then CSLA gene expression is different. Similar possibility occurs in varieties of Klangon and suweg. So in addition to genetic factors, environmental factors also play a role in determining glucomannan content An interesting phenomenon was shown by suweg. It was believed that suweg does not contain glucomannan, or if there is, it is very small. However, in this study it was proved that suweg also contains glucomannan, almost the same as the content of glucomannan of porang from Klangon. Further research is needed to ensure the content of glucomannan on suweg, because if the content of glucomannan high enough, it can be recommended as a source of high fiber food that is already well known and commonly eaten.

B.

Identification of CSLA gene

CSLA gene identification began with the isolation of genomic DNA. Originally it was planned to use only the tuber (corm) of suweg or porang. But it was then decided to isolate the leaves and spatha of porang from Klangon to see differences in the genes of different plant organs. The results showed differences in the size of isolated DNA (Fig.1.). DNA isolated from porang taken from different villages (Sumberbendo and Klangon) and different species of Amorphophallus (suweg) appeared to have different sizes. Additionally, for different parts of plants show different sizes of genomic DNA as well. The DNA sizes isolated from the leaves mostly larger than that isolated from the spatha or tuber and bulbil. Tuber and bulbil have the same DNA size. It has been investigated that A. muelleri Blume from Java has a high genetic diversity based on RAPD analysis [12]. RAPD is an analysis for investigating genome diversity without referring to a certain gene. So if the analysis recognized differences, the variation could be caused by differences in DNA sequence or differences in DNA size. Based on research done by Arumingtyas (unpublished) porang at East Java showed no variation based on trnL or rbcL sequence. This finding support the data in current research that differences in genomic DNA size is understandable. M 1 2 3 4 5 6

An interesting finding was shown by the existence of different number of band produced by porang and suweg. This explains the differences between the content of glucomannan in porang and suweg as shown by the analysis of glucomannan content. Based on this phenomenon it is proposed that CSLA gene has two isoforms that differ in size between suweg and porang.
M 1 2 3 4 5

700 bp 650 bp 600 bp

Fig. 2. PCR result using CSLA primer. M = marker; 1 = Leaf Sumber Bendo; 2= Flower spatha Klangon; 3 = Bulb Klangon; 4 = Corm Suweg; 5= negative control.

3 4 5 6

As mentioned before that the components of glucomannan is mannose and glucose. Mannose was formed with the help of the enzyme GDP-mannose pyrophosphorylase-D [2]-[4], while glucose is formed with the help of the enzyme GDP-D-glucose pyrophosphorylase [5]-[6]. Mannose and glucose subsequently formed into a chain with the help of glucomannan glucomannan synthase enzymes [7]. The existence of two isoforms in suweg may caused CSLA genes fail to produce glucomannan synthase which responsible in the formation of glucomannan chains, which is not happen in porang. Looking at the variation in PCR results of different parts of porang (leaf, flower spatha and bulbil), suggesting that genes on porang CSLA only exist in tissues that produces glucomannan as part of the flower, bulbil and tuber porang. This possibility needs to be further analyzed. Beside that the difference in the results of PCR of suweg and porang need to be analyzed further using DNA sequence analysis of those PCR bands. IV. CONCLUSION Glucomannan content between porang originating from different locations were not the same. This might be due to genetic and environmental factors. Suweg was found to have glucomannan. The differences of porang genomic DNA size have turned out to prove that there is genomic variation of Amorphophallus mueleri Blume investigated. CSLA genes were shown in porang and suweg. But, CSLA genes in porang are found only in the spatha flowers and the bulbs and not present in the leaves. It was detected that CSLA

Fig. 1. DNA genome of porang and suweg. M = marker; 1 = leaf of porang Sumber Bendo 1; 2 = leaf of porang Sumber Bendo 2; 3= flower spatha of porang Klangon 1; 4 = flower spatha of porang Klangon 2; 5= bulbil of porang Klangon; 6 = corm of Suweg 1.

PCR results for representatives of each piece of DNA porang varieties of Sumberbendo, Klangon and suweg (Fig. 2), indicated that suweg produces 2 CSLA band using primers designed based on the sequence of CSLA gene of Physcomitrella patens subsp. Patens. Flower spatha of porang from Klangon produce one band, while the leaves of Sumberbendo porang were not amplified. This may occur because the target gene was only positioned in the flowers and bulbs but not in the other parts of plants such as leaves.

genes of suweg differ from porang, these differences are likely to cause differences in the glucomannan produced. ACKNOWLEDGEMENT The author would like to thanks Dr. Serafinah Indriyani, Dr. Rodliyati Azrianingsih, Prof. Simon B Widjanarko for providing porang material, Fatchiyah, PhD for useful discussions. The research was supported by IMHERE grant REFERENCES
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