Analisis Polimorfisme Menggunakan Marka RAPD

Pengisian poin C sampai dengan poin H mengikuti template berikut dan tidak dibatasi jumlah kata atau halaman

namun disarankan seringkas mungkin. Dilarang menghapus/memodifikasi template ataupun menghapus

penjelasan di setiap poin.

Pada penelitian Keempat varietas ini mempunyai viabilitas benih yang bagus. Hal ini ditunjukkan dengan

nilai presentase daya kecambah varietas rewako F1 sebesar 85%, varietas Permata F1, Varietas Opal, dan Varietas

mutiara sebesr 90%. Menurut [1], hasil presentase daya kecambah ini merepresentasikan potensi viabilitas pada

benih. Viabilitas benih sendiri merupakan kemampuan benih untuk berkecambah dan selanjutnya berkembang

menjadi individu tanaman dewasa [2]. Benih yang memiliki viabilitas baik maka akan menghasilkan tanaman

dengan kualitas yang baik pula [2]. Presentase daya kecambah tersebut sesuai dengan literatur yang menyatakan

bahwa suatu benih memiliki daya kecambah yang baik bila memiliki presentase perkecambahan lebih dari 87%

[3].

Analisis Polimorfisme Menggunakan Marka RAPD

Analisis polimorfisme dilakukan untuk mengetahui variasi genetik dan pemetaan genetik suatu

organisme [4]. Langkah pertama analisis polimorfisme adalah menyediakan cetakan DNA. Cetakan DNA ini

diperoleh dari hasil ekstraksi DNA genomik 4 varietas tanaman tomat (Solanum lycopersicum). Selanjutnya,

cetakan DNA ini yang akan digunakan dalam proses amplifikasi DNA (PCR) dengan menggunakan beberapa

primer universal RAPD. Kualitas hasil ekstraksi DNA genomik selanjutnya dianalisis menggunakan

spektrofotometer. Kualitas ekstraksi ini menunjukkan kemurnian DNA yang dilihat dari nilai rasio A260/A280.

Hasil pengamatan menunjukkan bahwa ekstrak DNA genomik dari 4 varietas tersebut berada pada kisaran 1,8 –

2,08.

Nilai kemurnian kisaran 1,8 – 2,08 menunjukkan bahwa DNA memenuhi syarat kemurnian yang baik.

Hal ini sesuai dengan hasil penelitian [5], yang menyatakan bahwa ekstraks DNA genomik yang memiliki nilai

rasio A260/A280 antara 1,8 – 2,0 menunjukkan bahwa DNA telah memenuhi syarat kemurnian yang dibutuhkan

dalam analisis molekuler. DNA dikatakan berkualitas baik bila ekstral DNA tersebut bebas dari kontaminasi

seperti polifenol, protein, dan polisakarida [5]. Jika nilai rasio A260/A280 dibawah 1,8 maka DNA yang

diekstraksi tidak sesuai untuk pengamatan molekular karena terdapat kontaminasi polifenol dan kuantitas ekstrak

DNA rendah. Sedangkan jika nilai rasio A260/A280 diatas batas maksimum ( >2,0 ) maka DNA yang dihasilkan

terkontaminasi oleh protein, fenol atau senyawa lainnya dan RNA [6]. Kontaminasi RNA pada ekstraksi DNA

akan mengakibatkan munculnya pita RNA saat sampel diuji menggunakan gel agarose. Munculnya pita RNA

ditandai dengan banyaknya smirr pada pita DNA yang muncul [7].

Template DNA yang didapatkan selanjutnya digunakan dalam proses PCR untuk analisis polimorfisme.

Pada penelitian ini, analisis polimorfisme dilakukan dengan menggunakan marka molekular RAPD. Random

amplified polymorphic DNA (RAPD) dapat dilakukan untuk mendeteksi polimorfisme dengan menggunakan

sebuah primer tunggal RAPD yang akan menghasilkan Marka molecular [8]. Polimorfisme yang terjadi

ditunjukkan dengan ada atau tidaknya pita DNA. Analisis ini dilakukan untuk mengetahui tingkat keragaman

C. HASIL PELAKSANAAN PENELITIAN: Tuliskan secara ringkas hasil pelaksanaan penelitian yang telah

dicapai sesuai tahun pelaksanaan penelitian. Penyajian meliputi data, hasil analisis, dan capaian luaran

(wajib dan atau tambahan). Seluruh hasil atau capaian yang dilaporkan harus berkaitan dengan tahapan

pelaksanaan penelitian sebagaimana direncanakan pada proposal. Penyajian data dapat berupa gambar,

tabel, grafik, dan sejenisnya, serta analisis didukung dengan sumber pustaka primer yang relevan dan terkini.

genetik dari satu varietas dengan varietas lainnya. Analisis polimorfisme dalam penelitian ini menggunakan 5

primer universal, yaitu Primer S22, S119, OPV19, OPB18, dan S6 (Tabel 3.1). Hasil amplifikasi selanjutnya

dianalisis menggunakan elektroforesis gel agarose 1,5% (Gambar 4.1). Pita DNA yang terbentuk berkisar antara

300 bp – 1000 bp (Gambar 4.1)

Gambar 4.1 Hasil amplifikasi DNA menggunakan marka RAPD. A. Primer S22, B. Primer OPV19, C. Pimer

OPB 18, D. Primer S119 dan E. Primer S6; M = ladder, O = Opal, P = Permata F1, R = Rewako, M = Mutiara.

Tabel 4.1 tabel analisis polimorfisme menggunakan marka RAPD

No. Primer Sekuens Total

Lokus

Total

LP

%

Polimorfisme

Total

PT

Total

PP PIC

1 S22 AGTCAGCCAC 6 0 0% 12 0 0,75

2 OPV 19 GGGTGTGCAG 4 1 25% 7 1 0,80

3 OPB 18 CCACAGCAGT 3 0 0% 6 0 0,75

4 S119 TGCCGAGCTG 5 2 40% 7 2 0,86

5 S6 CTCACCGTCC 2 0 0% 4 0 0,75

Total 20 3 65% 36 3 3,91

Rata-Rata 4 0,6 13% 7,2 0,6 0,782

Keterangan : LP = Lokus Polimorfisme, PT = Pita yang terbentuk, PP = Pita Polimorfisme

Tabel 4.1 menunjukkan hasil perhitungan presentase polimorfisme 4 varietas tomat menggunakan

analisis marka RAPD. Nilai prosentase polimorfisme tanaman tomat berkisar 0% - 40%. Sedangkan nilai PIC

A B C

D E

yang didapat berkisar 0,75 – 0,86. Pada penelitian ini diperoleh total lokus sejumlah 22, total lokus polimorfisme

sejumlah 3, total pita yang terbentuk sejumlah 36 dan total pita polimorfisme sejumlah 3 buah.

Rentang nilai presentase polimorfisme yang dimiliki oleh beberapa primer menujukkan keragaman

genetik yang ada diantara keempat varietas uji berdasarkan marka RAPD. Semakin tinggi tingkat polimorfisme

maka semakin tinggi pula tingkat keragaman genetik suatu varietas [9]. Nilai PIC primer S22, OPB-18 dan S6

sebesar 0,75, OPV-19 sebesar 0,80 dan S119 sebesar 0,86 (Tabel 4.1). Nilai PIC tertinggi diperoleh primer S119

dan nilai PIC terendah diperoleh primer S22, OPB-18, dan S6. Pada penelitian [10] menunjukkan bahwa primer

S119 memiliki niali PIC sebesar 0,48. Hasil perhitungan nilai PIC pada penelitian ini lebih tinggi dibandingkan

dengan hasil penelitian [10] yang memperoleh rentang nilai PIC 0,31 - 0,50. Penelitian tersebut juga menganalisis

keragaman genetik beberapa varietas tomat di Cina. Nilai PIC dijadikan sebagai standar polimorfisme suatu lokus

antara genotip dengan menggunakan informasi jumlah alel [11] serta sebagai untuk menentukan tingkat informatif

suatu marka molekuler (primer). Nilai PIC dibagi menjadi tiga kategori yaitu PIC > 0,60 = Informatif tinggi,

kemudian 0,3 > 0,59 = cukup informatif dan PIC < 0,3 = Kurang Informatif [12]. Berdasarkan hasil yang

diperoleh, semua primer RAPD memiliki nilai PIC ≥ 0,7, hal ini menunjukkan bahwa keseluruhan primer RAPD

yang digunakan termasuk dalam ketegori marka molekuler yang sangat informatif.

Data pita DNA polimorfisme yang telah diperoleh, selanjutnya digunakan sebagai dasar untuk

merekonstruksi pohon filogeni. Hal ini dilakukan untuk melihat kekerabatan berdasarkan kesamaan karakter

antara varietas satu dengan varietas yang lain. Analasis filogenetik direpresentasikan sebagai sistem percabangan,

seperti diagram pohon yang dikenal sebagai pohon filogenetik. Data hasil skoring kemudian dianalisis

menggunakan program UPGMA (Unweighted Pair Group Method with Arithmetic) melalui software MVSP.

Hubungan kekerabatan antara varietas tomat (Solanum lycopersicum) dapat diketahui dari indeks koefisien

kesamaan jaccard. Indeks koefisien kesamaan jaccard terbentang dari 0 sampai 1. Indeks kesamaan jaccard ini

berfungsi untuk melihat kesamaan dan membandingkan antara satu individu dengan individu lainnya. Rentang

kesamaan yang digunakan dari 0% hingga 100%. Semakin tinggi presentasenya, semakin mirip kedua populasi

tersebut.

Gambar 4.2 Dendogram hubungan kekerabatan tanaman tomat (Solanum lycopersicum) berdasarkan marka

RAPD.

Dendogram pada gambar 4.2 menunjukkan bahwa terdapat 3 klaster yaitu klaster I adalah varietas

Rewako dengan similaritas sebesar 0, Klaster II Permata dengan similaritas sebesar 0 serta klaster II adalah

varietas Mutiara dan Opal dengan similaritas sebesar 0,818. Nilai similaritas 0,818 atau sama dengan 81%

menunjukkan bahwa varietas mutiara dan opal memiliki hubungan kekerabatan yang dekat. Nilai similaritas pada

varietas Mutiara dan Opal hampir mendekati nilai similaritas pada penelitian [9]. Penelitian [9] menunjukkan

bahwa dari 14 varietas tanaman tomat (Solanum lycopersicum) lokal di Nigeria memiliki nilai similaritas sebesar

100%. Nilai similaritas sebesar 100% menunjukkan bahwa pada semua varietas tanaman tomat sama.

Pengaruh Logam berat Pb terhadap Morfologi tanaman Solanum lycopersicum

Logam berat timbal (Pb) merupakan jenis logam berat yang termasuk kedalam logam berat paling beracun

dan tidak memiliki peran dalam sistem biologis [13]. Timbal (Pb) dapat memberikan pengaruh salah satunya pada

morfologi tanaman [14]. Salah satu parameter morfologi yang terdampak karena adanya cekaman logam berat

adalah sistem perakaran dan tinggi tanaman [15-16]. kerusakan sel akar, menurunnya panjang akar dan klorosis

pada daun [17] gejala ini disebabkan oleh ketidak seimbangan air, nutrisi mineral yang terganggu, atau kerusakan

enzim [18-19].

Pada penelitian ini digunakan Pb dengan konsentrasi 0 ppm, 75 ppm, 150 ppm, dan 300 ppm dengan lama

cekaman selama 32 hari. Cekaman logam berat Pb yang diberikan pada 4 varietas tanaman tomat (Solanum

lycopersicum) selama 32 hari mempengaruhi panjang akar tanaman tomat. Gambar 4.3 menunjukkan hasil

perhitungan rata – rata panjang akar tanaman tomat dimana panjang akar tanaman tomat yang diberikan cekaman

lebih pendek dibandingkan dengan kontrol. Perbandingan rata – rata panjang akar ditunjukkan pada (Gambar 4.3)

Keterangan : A0: Opal 0 ppm, A1: Opal 75 ppm, A2: Opal 150 ppm, A3: Opal 300 ppm, B0: Permata 0 ppm, B1:

Permata 75 ppn, B2: Permata 150 ppm, B3: Permata 300 ppm, C0: Mutiara 0 ppm, C1: Mutiara 75 ppm, C2:

Mutiara 150 ppm, C3: Mutiara 300 ppm, D0: Rewako 0 ppm, D1: Rewako 75 ppm, D2: Rewako 150 ppm, D3:

Rewako 300 ppm.

Pada Gambar 4.3 dapat dilihat bahwa dari semua perlakuan logam berat Pb tanaman tomat yang memliki

rata – rata panjang paling rendah adalah varietas Opal yang diberi perlakuan Pb dengan konsentrasi 75 ppm yaitu

sebesar 3 cm. Selanjutnya disusul oleh varietas Rewako 300 ppm, varietas Permata 75 ppm, dan varietas Mutiara

150 ppm dengan rerata panjang akar masing-masing sebesar 3,24 cm, 3,40 cm, dan 4,10 cm. Hal ini menunjukkan

bahwa pada setiap konsentrasi logam berat akan memberikan penurunan pada panjang akar (Gambar 4.4). Pada

penelitian Yilmaz et al., (2010) dan Akinci et al., 2010 menunjukkan bahwa tanaman S. Melongena dan Solanum

lycopersicum yang tercekam logam berat Pb konsentrasi 75 ppm, 150 ppm dan 300 ppm memiliki panjang akar

lebih pendek dibandingkan dengan kontrol.

Gambar 4.4 foto akar Solanum lycopersicum pada kontrol, Pb konsentrasi 75 ppm, 150 ppm dan 300ppm; (A)

Varietas Permata F1, (B) Varietas Opal, (C) Varietas Mutiara, (D) Varietas Rewako.

Nilai rata – rata panjang akar kontrol lebih tinggi dibandingkan dengan nilai rata – rata panjang akar

perlakuan disebabkan karena sebagian besar logam berat Pb yang diserap oleh tanaman dari tanah akan tetap

terakumulasi lebih banyak pada akar dan sebagian kecil akan dipindahkan ke batang dan daun [20]. Lebih banyak

logam berat Pb yang terakumulasi pada akar akan menyebabkan pengurangan panjang akar. Hal ini disebabkan

karena akumulasi logam berat didalam akar akan megurangi laju pembelahan sel di tingkat mitosis pada tahap

metafase dalam zona meristematik akar [21]. Menurut [14], akar tanaman yang berada pada kondisi tercekam

logam berat Pb akan mensintesis callose. Callose yang disintesis ini akan menghambat penyerapan Pb. Selain itu,

callose yang terbentuk ini secara bersamaan akan menghambat transportasi molekul lain seperti nutrisi yang

masuk kedalam sel yang akan membuat pertumbuhan akar terhambat. Selain itu, akumulasi logam berat di dinding

sel dan ikatannya dengan karbohidrat akan menurunkan plastisitas dinding sel. Hal ini akan menghambat

pembelahan sel dan elongasi sel yang selanjutnya berdampak pada berkurangnya ukuran dari sel yang sedang

berkembang [18].

Penurunan rata – rata panjang akar pada tanaman yang tercekam dapat disebabkan oleh beberapa faktor

diantaranya pH. Pada penelitian ini juga dilakukan pengukuran pH tanah. Hasil dari pengukuran pH tanah

menunjukkan bahwa pH tanah menjadi berubah menjadi cenderung asam setelah pemberian perlakuan Pb dengan

beragam konsentrasi yang berbeda. Perubahan pH pada tanah menjadi indikator bahwa terdapat akumulasi logam

berat Pb yang selanjutnya akan diserap oleh akar tanaman [22]. Perubahan pH menjadi asam akan mempengaruhi

pertumbuhan tanaman [23]. Adanya perubahan pH pada tanah disebabkan oleh pelepsan hidroksil (OH-) dan

pengambilan proton (H+) oleh akar yang merupakan komponen utama dalam alkalinisasi tanah. Akar tanaman

akan melepaskan H+ saat akar terlalu banyak menyerap kation daripada anion, karena untuk mempertahankan

keseimbangan muatannya [24]. Penyerapan (OH-) dan pelepasan (H+) akan menyebabkan perubahan dalam

aktivitas pompa proton (H+ ATPase) yang akan menyebabkan terhambatnya pertumbuan akar [25].

Berdasarkan uji statistik menunjukkan bahwa konsentrasi logam berat Pb, Varietas tanaman tomat dan

interaksi antar keduanya memiliki pengaruh terhadap tinggi tanaman tomat. Hal ini ditunjukkan dengan nilai

A B

C D

signifikan (p = 0,000). Tabel 4.2 menunjukkan hasil bahwa pada semua varietas yang diberikan cekaman logam

berat Pb pada konsentrasi 300 ppm memiliki nilai rata – rata tinggi tanaman paling tinggi dibandingkan denhan

konsentrasi lain dan kontrol.

Logam berat yang dibawa menuju batang dan daun akan terakumulasi dan memberikan efek pada

tanaman. Pada penelitian ini pengukuran tinggi tanaman dilakukan pada hari ke 30 pemberian cekaman logam

berat Pb dan dibandingkan tinggi tanaman antara kontrol dan diberikan cekaman logam berat Pb. Hasil analisis

rata – rata tinggi tanaman ditunjukkan pada (Tabel 4.2).

Tabel 4.2 Tabel Rata – rata tinggi tanaman (cm) dan nilai St.Dev

Varietas/

PPM Opal Permata Mutiara Rewako

0 ppm 39,00 ± 2,09 a 52,70 ± 2,68 ad 41,20 ± 1,92 ab 48,60 ± 1,60 ac

75 ppm 40,86 ± 3,49 ba 57,30 ± 1,44 bd 48,20 ± 2,80 b 56,30 ± 2,25 bc

150 ppm 45,72 ± 2,43 ba 61,40 ± 1,52 bd 50,24 ± 1,60 b 50,50 ± 0,00 bc

300 ppm 45,04 ± 3,70 ca 64,00 ± 3,22 cd 50,40 ± 0,22 cb 53,50 ± 2,12 c

Keterangan: Nilai yang diikuti oleh huruf yang berbeda pada tiap kolom varietas tomat menujukkan adanya

berbeda nyata pada uji Tukey dengan taraf α=0,05.

Pada penelitian ini menunjukkan hasil bahwa tanaman tomat memiliki respon yang berbeda terhadap

adanya cekaman logam berat. Nilai rata – rata tinggi tanaman tomat tercekam Pb lebih tinggi dibandingkan dengan

kontrol. Adanya cekaman logam berat secara berlebihan selain akan memberikan dampak negatif pada tanaman

juga dapat memicu mekanisme perlawanan atau perlindungan diri oleh tanaman terhadap keberadaan logam berat.

Salah satu mekanisme perlawanan atau perlindungan diri pada tanaman adalah melalui sintesis fitokhelatin (PCs)

dan metallothionein (MTs), dimana fitokhelatin (PCs) merupakan suatu molekul yang memiliki fungsi primer

dalam proses detoksifikasi, dan metallothionein (MTs) yang bertindak dalam translokasi logam [26]. Fitokhelatin

disintesis dari glutathione atau homolognya secara enzimatik melalui rekasi katalis oleh fitokhelatin sintase, yaitu

enzim yang diaktivasi oleh keberadaan logam berat (termasuk Pb). Fitokhelatin memiliki komponen sistein yang

tinggi sehingga mudah menciptakan kompleks dengan logam toksik [27].

Cekaman logam berat juga akan memicu tanaman untuk memproduksi antioksidan berupa katalase dan

peroksidase [26]. Katalase dan peroksidase merupakan beberapa enzim kunci yang mempertahankan sel ketika

melawan cekaman oksidatif yang disebabkan oleh ROS (Reactive Oxygen Spexies) seperti H2O2 . Degradasi H2O2

menjadi air dan oksigen akan dibawa oleh katalase menuju peroksisom atau oleh peroksidase menuju vakuola,

dinding sel dan sitosol, sehingga tidak akan mengganggu metabolisme tanaman. Sehingga tanaman yang toleran

terhadap pemaparan logam berat dalam konsentrasi tinggi akan memperlihatkan peningkatan aktivitas katalase

dan peroksidase ketika terpapar logam berat [28].

Tingginya nilai rata – rata tinggi tanaman juga bisa disebabkan oleh adanya akumulasi Pb pada akar dan

mobilitas Pb menuju batang rendah. Hal ini disebabkan oleh kuatnya afinitas pengikat Pb pada dinding sel akar

dan membentuk endapan kristal, sehingga logam tersebut akan banyak tertahan di akar. Logam berat Pb diserap

oleh rambut akar dan disimpan pada dinding sel dalm konsentrasi yang cukup tinggi. Transportasi Pb ke bagian

batang dan daun sangat terbatas, yaitu hanya sekitar 3%, sehingga Pb akan terakumulasi dalam jumlah yang cukup

besa di dalam akar. Dinding sel dan vakuola merupakan komponen utama yang bertanggungjawab dalam

penyimpanan Pb pada tumbuhan [29]. Akumulasi Pb di dinding sel dan vakuola diketahui merupakan mekanisme

perlindungan tanaman untuk menjaga agar konsentrasi ion toksik di sitoplasma rendah [30]. Hal ini menunjukkan

bahwa transportasi logam berat Pb tidak anya secara pasif di dinding sel atau ruang intersellular, tetapi juga

ditransporkan ke sitoplasma [30].

D. STATUS LUARAN: Tuliskan jenis, identitas dan status ketercapaian setiap luaran wajib dan luaran

tambahan (jika ada) yang dijanjikan. Jenis luaran dapat berupa publikasi, perolehan kekayaan intelektual,

hasil pengujian atau luaran lainnya yang telah dijanjikan pada proposal. Uraian status luaran harus didukung

dengan bukti kemajuan ketercapaian luaran sesuai dengan luaran yang dijanjikan. Lengkapi isian jenis

luaran yang dijanjikan serta mengunggah bukti dokumen ketercapaian luaran wajib dan luaran tambahan

melalui Simlitabmas.

Luaran dari penelitian ini akan dipublikasikan di seminar Internasional IBOC (International Biology Conference).

Adapun seminar tersebut akan diselenggarakan pada 17 Oktober 2020. Judul dari makalah yang akan

dipresentasikan adalah Genetic Diversity and Morphological Response of Local Tomato Varieties (Solanum

Lycopersicum) under Pb Stress. Selain itu, jurnal internasional dengan judul An ethnobotanical study of

medicinal plants used by the Tengger tribe in Ngadisari village, Indonesia telah terbit di jurnal Plos One

(Scopus indexed, Q1).

E. PERAN MITRA: Tuliskan realisasi kerjasama dan kontribusi Mitra baik in-kind maupun in-cash (untuk

Penelitian Terapan, Penelitian Pengembangan, PTUPT, PPUPT serta KRUPT). Bukti pendukung realisasi

kerjasama dan realisasi kontribusi mitra dilaporkan sesuai dengan kondisi yang sebenarnya. Bukti dokumen

realisasi kerjasama dengan Mitra diunggah melalui Simlitabmas.

Penelitian ini tidak memiliki mitra.

F. KENDALA PELAKSANAAN PENELITIAN: Tuliskan kesulitan atau hambatan yang dihadapi selama

melakukan penelitian dan mencapai luaran yang dijanjikan, termasuk penjelasan jika pelaksanaan penelitian

dan luaran penelitian tidak sesuai dengan yang direncanakan atau dijanjikan.

Kendala utama penelitian ini adalah pemberlakuan kegiatan eksperimental laboratorium terbatas. Hal ini

dikarenakan adanya pandemi covid-19 yang mengharuskan seluruh aktivitas laboratorium mengikuti protokol

ketat covid-19. Hal ini sedikit banyak membuat penelitian sedikit memerlukan ekstra waktu.

G. RENCANA TAHAPAN SELANJUTNYA: Tuliskan dan uraikan rencana penelitian di tahun berikutnya

berdasarkan indikator luaran yang telah dicapai, rencana realisasi luaran wajib yang dijanjikan dan

tambahan (jika ada) di tahun berikutnya serta roadmap penelitian keseluruhan. Pada bagian ini

diperbolehkan untuk melengkapi penjelasan dari setiap tahapan dalam metoda yang akan direncanakan

termasuk jadwal berkaitan dengan strategi untuk mencapai luaran seperti yang telah dijanjikan dalam

proposal. Jika diperlukan, penjelasan dapat juga dilengkapi dengan gambar, tabel, diagram, serta pustaka

yang relevan. Jika laporan kemajuan merupakan laporan pelaksanaan tahun terakhir, pada bagian ini dapat

dituliskan rencana penyelesaian target yang belum tercapai.

Tahap selanjutnya akan lebih difokuskan pada aspek penelusuran literatur terkait respon fisiologi varietas lokal

tomat meliputi kadar prolin, kandungan klorofil, dan molekular yang akan dianalisa secara deskriptif kuantitatif.

beberapa gen yang akan ditelaah antara lain adalah gen-gen yang teribat dalam protein folding (LEMT-1, LEMT-

2, LEMT-3, LEMT-4, LEHsp90-1), biosintesis fitohormon (NCED 2/3, PIN1, EIN 2) dan metabolit antioksidan

(P5CS1, GSH).

H. DAFTAR PUSTAKA: Penyusunan Daftar Pustaka berdasarkan sistem nomor sesuai dengan urutan

pengutipan. Hanya pustaka yang disitasi pada laporan kemajuan yang dicantumkan dalam Daftar Pustaka.

1. Khan, N., Kazmi, R.H., Willems, L.A.J., van Heusden, A.W., Ligterink, W., Hilhorst, H.W.M. 2012.

Exploring the Natural Variation for Seedling Traits and Their Link with Seed Dimensions in Tomato.

PloS one 7: e43991.

2. Shaban, M. 2013. Study on some aspects of seed viability and vigor. International journal of Advanced

Biological and Biomedical Research. Vol.1 Issue 12: 1692-1697.

3. Peñaloza, P., & Durán, J.M. (2015). Association between biometric characteristics of tomato seeds and

seedling growth and development. Electronic Journal of Biotechnology, 18, 267-272.

4. Dubiley, S., Kirillov, E., Mirzabekov, A. 1999. Polymorphism analysis and gene detection by

minisequencing on an array of gel-immobilized primers. Oxford university Press. Nucleic Acid

Research. Vol 27;18.

5. Desjardins P, Conklin D. 2010. NanoDrop microvolume quantitation of nucleic acids. J Vis Exp.

6. Sambrook J, Russell DW .2001. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor

Laboratory Press, New York.

7. Wang, X., Xiao, H., Zhao, X., Li, C., Ren, J., Wang, F., Pang, L. 2012. Isolation of high-quality DNA

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RESEARCH ARTICLE

An ethnobotanical study of medicinal plants

used by the Tengger tribe in Ngadisari village,

Indonesia

Nurul JadidID*, Erwin Kurniawan, Chusnul Eka Safitri Himayani, Andriyani,

Indah PrasetyowatiID, Kristanti Indah Purwani, Wirdhatul Muslihatin, Dewi Hidayati, Indah

Trisnawati Dwi Tjahjaningrum

Department of Biology, Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia

* [email protected]

Abstract

The people of Tengger, Indonesia have used plants as traditional medicine for a long time.

However, this local knowledge has not been well documented until recently. Our study aims

to understand the utilization of plants in traditional medicine by the people of Tengger, who

inhabit the Ngadisari village, Sukapura District, Probolinggo Regency, Indonesia. We con-

ducted semi-structured and structured interviews with a total of 52 informants that repre-

sented 10% of the total family units in the village. The parameters observed in this study

include species use value (SUV), family use value (FUV), plant part use (PPU), and the rela-

tive frequency of citation that was calculated based on fidelity level (FL). We successfully

identified 30 species belonging to 28 genera and 20 families that have been used as a tradi-

tional medicine to treat 20 diseases. We clustered all the diseases into seven distinct cate-

gories. Among the recorded plant families, Poaceae and Zingiberaceae were the most

abundant. Plant species within those families were used to treat internal medical diseases,

respiratory-nose, ear, oral/dental, and throat problems. The plant species with the highest

SUV was Foeniculum vulgare Mill. (1.01), whereas the Aloaceae family (0.86) had the high-

est FUV. Acorus calamus L. (80%) had the highest FL percentage. The leaves were identi-

fied as the most used plant part and decoction was the dominant mode of a medicinal

preparation. Out of the plants and their uses documented in our study, 26.7% of the medici-

nal plants and 71.8% of the uses were novel. In conclusion, the diversity of medicinal plant

uses in the Ngadisari village could contribute to the development of new plant-based drugs

and improve the collective revenue of the local society.

Introduction

The interaction between humans and plants has been long described as one of the factors influ-

encing human civilization, especially in medicinal fields [1]. Documentation of the medicinal

use of plants through ethnobotanical studies enables the development of contemporary drugs

and treatments as well as for plant conservation [2, 3]. Many ethnobotanical studies around

PLOS ONE

PLOS ONE | https://doi.org/10.1371/journal.pone.0235886 July 13, 2020 1 / 16

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OPEN ACCESS

Citation: Jadid N, Kurniawan E, Himayani CES,

Andriyani , Prasetyowati I, Purwani KI, et al. (2020)

An ethnobotanical study of medicinal plants used

by the Tengger tribe in Ngadisari village, Indonesia.

PLoS ONE 15(7): e0235886. https://doi.org/

10.1371/journal.pone.0235886

Editor: Khawaja Shafique Ahmad, University of

Poonch Rawalakot, PAKISTAN

Received: August 18, 2019

Accepted: June 23, 2020

Published: July 13, 2020

Copyright: © 2020 Jadid et al. This is an open

access article distributed under the terms of the

Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevant data are

within the manuscript and its Supporting

Information files.

Funding: This work is financially supported by

Kementerian Riset Teknologi dan Pendidikan Tinggi

Republik Indonesia (Contract no. 890/PKS/ITS/

2019 and contract no. 1204/PKS/ITS/2020). NJ

received grant from Ministry of Research,

Technology and Higher Education of the Republic

of Indonesia. The funders had no role in study

http://orcid.org/0000-0001-9387-4919

http://orcid.org/0000-0001-8637-0851

https://doi.org/10.1371/journal.pone.0235886

http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0235886&domain=pdf&date_stamp=2020-07-13








http://creativecommons.org/licenses/by/4.0/

the world, including in Indonesia, report the use of herbal plants for the healing process,

which has been in use for several generations in their respective societies [4, 5]. Though the

cultural diversity in Indonesia contributes to the extensive this traditional knowledge [6],

access to this is limited. Traditional knowledge is usually passed on orally and often person-

specific [7]. Therefore, the knowledge is often owned by tribal leaders, village heads, elders,

heads of kampung (small village), or traditional healers in the particular community or tribe

[8].

Indonesia has around 40,000 different plant species, of which approximately 6,000 are used

for traditional healing processes [4], especially in certain tribal areas including Bromo Tengger

Semeru National Park (BTSNP) [9]. BTSNP is designated as a national park because of its fas-

cinating vegetation (about 600 floral species) and is home to the unique Tengger tribe. Some

plants have been cultivated for daily consumption and trading whereas others are naturally

found and used for particular purposes such as tribal ceremonies and medicinal uses [9]. Peo-

ple of Tengger are distributed in buffer zone villages around the BTSNP including Ngadisari

village [10].

The Tengger people use plants from the BTSNP for traditional ceremonies [11], as well as

medicinal applications [12], industrial materials, food sources, and building materials in some

buffer village areas [13]. However, there are no reports regarding the ethnobotanical aspect of

medicinal plants used in these buffer village areas in the BTSNP by the Tengger people. The

present study documents the medicinal plant species and traditional knowledge of the Tengger

tribe who inhabit the Ngadisari village in the BTSNP, Indonesia.

Materials and methods

Study area

This study was carried out in Ngadisari village, which belongs administratively to the Sukapura

district in Probolinggo Region of the Republic of Indonesia. It is located at 7˚ 55’ 18” S 112˚

57’ 21” E around the Bromo Tengger Semeru National Park (BTSNP) (Fig 1). Ngadisari village

is situated at an altitude of 1800–1950 meters above sea level. The total area of the study com-

prised 4,993 km2. Like other regions in Indonesia, Ngadisari village has only two seasons; dry

and rainy. The rainy season spans the months of November-May, whereas the dry season

spans June-October. The present study was conducted from 2018–2019. Most inhabitants

belonged to the Tengger tribe and rely on agriculture. According to the Indonesian Statistics

Bureau (BPS) data, the Ngadisari village has a population of 1,543 inhabitants or about 507

family units (households) [14]. The population consists of 742 males and 801 females. The

Welsh onion (Allium fistulosum L.), potato (Solanum tuberosum L.), cabbage (Brassica oleraceaL.), carrot (Daucus carota L.) and corn (Zea mays L.) are examples of plants that contribute to

the income of these communities [9].

Data collection

This study was authorized (SK No. 091650/IT2.VII/HK.00.02/2018) by the Institute of

Research and Community Service (LPPM) of the Institut Teknologi Sepuluh Nopember (ITS),

Surabaya, Indonesia. Verbal informed consent was obtained from each informant before con-

ducting the interview process.

Data collection was obtained through semi-structured and structured interviews with infor-

mants who knew or used plants as medicine. This technique is commonly used in ethnobotan-

ical studies [16]. Interviews were conducted with selected informants including about 10% of

the total heads of family units (52 informants) to determine and explore the traditional knowl-

edge regarding the utilization of medicinal plant species, their usefulness, the utilized part,

PLOS ONE Ethnobotany of medicinal plants used by Tengger tribe


design, data collection and analysis, decision to

publish, or preparation of the manuscript.

Competing interests: The authors have declared

that no competing interests exist.


mode of preparation, or method of processing the plants. All of the head family units were

males since norms, values, and local wisdom are based on patriarchal culture [17]. The age of

the informants ranged from 25 to more than 45 years, where four were between the ages of

Fig 1. Location of the study area. Ngadisari village (A, red color) is located within the Bromo Tengger Semeru National Park Indonesia. This figure is similar but not

identical to the original image obtained from [15] under a CC BY license and is used for illustrative purposes only.

https://doi.org/10.1371/journal.pone.0235886.g001





25–30, sixteen were ranging from 31–35, eighteen were between 36–40, nine were ranging

from 41–45, and five informants were older than 45 years. The interview activities were carried

out in their entirety using a questionnaire. Informant selection was based on the Snowball

Sampling technique, by determining the key person. A key-person is one who possesses strong

power within society. The subsequent informants are determined by the direction of the previ-

ous respondents.

Taxonomical identification and herbarium

Taxonomical identification was conducted to verify the samples that were raised during the

interviews. An herbarium was also prepared to obtain dry specimens supporting the taxonom-

ical identification. However, the herbarium method was used only for unknown species. Photo

documentation and herbarium of medicinal plants were then identified by Christin Risbandini

under the laboratory of plant bioscience and biotechnology, Institut Teknologi Sepuluh

Nopember, Indonesia using key dichotomy and some references [18, 19].

Disease classification and grouping

Diseases that commonly occur in the Indonesian region were grouped into seven categories

including gastrointestinal disorders (GI) (diarrhea, nausea, vomiting, stomach ache, gastric

problems, loss of appetite, colic, flatulence, dysentery); dermatological diseases (DO) (skin

burns, skin spots, skin rashes, boils, cut, wounds, hair problems, ectoparasites); urogenital and

gynecological problems (UGP) (sexual problems including frigidity, lack of libido, infertility,

gonorrhea, diuretic, aphrodisiac, menstrual disorders); skeletomuscular disorders (SD); inter-

nal medical diseases (IM) (diabetes, cancers, and tumors, hypertension, piles/hemorrhoids);

respiratory-nose, ear, oral/dental, throat problems (RT) (asthma, nose bleeding, sinusitis, ear-

ache, throat shore, dental problems); and others (OT) (motion sickness).

Data analysis

Fidelity Level (FL). The relative frequency of citation was calculated using the fidelity

level (FL) formula according to Friedman et al. [20] and Ouedraogo et al. [21]. FL is the per-

centage of informants who claim to use certain plant species for particular healing processes.

This reflects the preference of people for a specific plant species in a particular medicinal treat-

ment. It was calculated using the following equation:

FL %ð Þ ¼NpN

x 100 ð1Þ

Where Np is the number of informants who mentioned or claimed the use of plant species

for a particular healing process/medicinal treatment. N is the total number of informants who

cited the plant species for various kinds of medicinal treatment.

Species Use Value (SUV). SUV signifies the value of a medicinal plant species used by the

people from Ngadisari village. It is calculated as the sum of the informant species use values

(UVis) for a particular medicinal species divided by the total number of informants (Ni). The

SUV was calculated according to Hoffman and Gallaher [22] as follows:

SUV ¼P

UVisðniÞ

ð2Þ

Family Use Value (FUV). FUV was calculated as described by Phillips and Gentry [23],

signifying the use value of a given plant family that is used as medicine by the people from




Ngadisari village. The calculation follows the below equation:

FUV ¼P

UVsðnsÞ

ð3Þ

Where ∑UVs represents the sum of the use values for all species belonging to a particular

family divided by the total number of species in the same family.

Plant Part Value (PPV). The plant part value is presented as the percentage of utilized

parts of plants (stem, leaves, root, fruit, bark, and flower) that are used as medicinal biore-

sources. The PPV is calculated according to Gomez-Beloz [24] as follows:

PPV %ð Þ ¼P

RUðplant partÞP

RUx 100 ð4Þ

Where ∑RU(plant part) and ∑RU represent the sum of the cited plant parts and the total num-

ber of cited uses for a given plant, respectively.

Results and discussion

Utilization of plant species as traditional medicine by Tengger tribe in

Ngadisari village

The people of Tengger receive their knowledge of traditional medicine from their ancestors.

This knowledge is inherited and subsequently preserved across generations [12]. We found 30

plant species that are used in traditional medicine. Among them, eight plants (26.7%) were

recorded for the first time, compared with the previous study [9, 11, 12]. They were Mandevillasanderi (Hemsl.) Woodson, Jatropha curcas L., Cymbopogon nardus (L.) Rendle), Microsorumbuergerianum (Miq.) Ching., Paederia foetida L., Solanum muricatum Ait., Zingiber zerumbet(L.) Sm., and Senna alata (L.) Roxb. Also, different medicinal uses for known plants (71.8%)

were observed in the present study, compared to the study conducted by Batoro [9] (Table 1).

The highest number of species in one category was found in the category of IM with 12 spe-

cies, followed by nine species in RT and six species in DD (Table 1). We also observed that five

species found in this study were used to treat more than one disease in distinct categories. For

instance, fennel, locally named Adas (Foeniculum vulgare Mill.) has been used to treat urti-

caria/hives (DD), cough (RT), and to overcome motion sickness (OT). Betelvine (Piper betleL.) has also been used for more than one disease including leucorrhoea (UGP), hives or urti-

caria (DD), and worm disease (GI). This demonstrated that the use value of these species is

quite high compared with that of other medicinal plants [22].

Species and family use value

Species use value demonstrates the value of a medicinal plant species used by the people from

Ngadisari village. Our results revealed that the SUV of the reported plants varied from 0.01 to

1.01 (Fig 2). Five species showed the highest SUV: Foeniculum vulgare Mill. (1.01), Aloe vera(L.) Burm. f. (0.86), Acorus calamus L. (0.8), Apium graveolens L. (0.76), and Allium fistulosumL. (0.71). Previous studies also demonstrated that fennel is frequently used as medicinal plants

in Indonesia [12, 25] and is abundantly present in this region [9]. Our data showed that F. vul-gare is categorized as a plant used to treat dermatological problems (DO). People of Tengger

inhabiting Ngadisari village use F. vulgare to treat urticaria, hives, or itching. Our results are

also in accordance with other studies that revealed F. vulgare as a traditional medicine for peo-

ple suffering from itching or other dermatitis problems [26].




Table 1. Disease categories, health-related problems, and medicinal plants used in Ngadisari village.

No Disease Categories Specified disease

name

Plant family Plant species Common name Local name Plant part

used

Mode of

preparation

1 Internal medical diseases Hypertension Apiaceace Apium graveolens L. Celery Seledri Leaves Eaten raw,

decoction

Solanaceae Physalis angulata L. Cutleaf ground

cherry

Keciplukan Leaves Decoction

Solanaceae Solanum muricatum Ait. Pepino dulce,

sweet cucumber

Buah Melodi Fruit Eaten raw

Zingiberaceae Zingiber zerumbet (L.) Sm. Bitter ginger Lempuyang Rhizome Eaten raw

Fever Acoraceae Acorus calamus L. Sweet flag,

calamus

Dringu Leaves Pounded

Liliaceae Allium cepa L. Onion Bawang merahtengger

Bulb Burned

Poaceae Cymbopogon nardus (L.)

Rendle

Citronella grass Serai Leaves Squeezed

Poaceae Saccharum officinarum L. Sugarcane Tebu merah Stem Burned

Zingiberaceae Curcuma domestica Val. Curcuma Kunyit Rhizome Shredded

Nose bleeding Asteraceae Artemisia vulgaris L. common

wormwood

Ganjan Leaves Rolled up

Hemorrhoid Myrtaceae Psidium guajava L. Guava Jambu klutuk Leaves Pounded

Solanaceae Physalis angulata L. Cutleaf Ground

Cherry

Keciplukan Leaves Decoction,

pounded

Clusiaceae Garcinia mangostana L. Mangosteen Manggis Stem bark Burned

2 Urogenital and

gynecological problems

Leucorrhea Piperaceae Piper betle L. Betelvine Sirih Leaves Decoction

Rubiaceae Paederia foetida L. Stinkvine Kesimbukan Leaves Decoction

3 Dermatological diseases Hair problems Arecaceae Cocos nucifera L. Coconut Kelapa Fruit Decoction

Aloaceae Aloe vera (L.) Burm. f. Barbados aloe Lidah Buaya Leaves Smeared

Urticaria/hives Apiaceace Foeniculum vulgare Mill. Fennel Adas Leaves Decoction,

Pounded

Piperaceae Piper betle L. Betelvine Sirih Leaves Decoction

Polypodiaceae Microsorum buergerianum(Miq.) Ching.

Microsorum Pangotan,

paduka ajiLeaves Decoction

Ringworm Fabaceae Senna alata (L.) Roxb. Candle bush Ketepeng Leaves Pounded,

decoction

Skin burn Aloaceae Aloe vera (L.) Burm. f. Barbados aloe Lidah buaya Leaves Smeared

4 Respiratory-nose, ear, oral/

dental, throat problems

Cough Liliaceae Allium fistulosum L. Welsh onion Bawang prei Leaves Burned

Apiaceace Foeniculum vulgare Mill. Fennel Adas Leaves Decoction

Rutaceae Citrus aurantium L. Lime Jeruk Nipis Fruit Squeezed

Zingiberaceae Zingiber officinale Rosc. Ginger Jahe Rhizome Pounded,

decoction

Zingiberaceae Kaempferia galanga L. Chinese ginger,

aromatic ginger

Kencur Rhizome Burned

Mouth ulcer,

sprue

Euphorbiaceae Jatropha curcas L. Jatropha Jarak Pagar Stem Smeared

Asthma Poaceae Cymbopogon nardus (L.)

Rendle

Citronella grass Serai Leaves Decoction

Heatiness Poaceae Imperata cylindrica (L.) P.

Beauv.

Cogon grass Alang-alang Leaves Decoction

Eye Irritation Apocynaceae Mandevilla sanderi(Hemsl.) Woodson

Brazilian jasmine BungaTerompet

Gum Dropped

(Continued)




Similar medicinal uses of Aloe vera (L.) Burm. f., Acorus calamus L., Apium graveolens L.,

and Allium fistulosum L. have been reported in previous ethnobotanical studies. For example,

Reimers et al. [27] and Salehi et al. [28] reported the use of Aloe vera to treat hair problems.

Meanwhile, the use of Acorus calamus L. to treat fever has been reported by Rajput et al. [29].

Also, A. graveolens and A. fistulosum L. have been used by traditional Chinese and Indonesian

people to reduce blood pressure and cough, respectively [30, 31]. Finally, nine species were

reported to have low SUV (0.01) in the present study (Fig 2). High or low SUV may be due to

extensive or minimum ethnobotanical uses of the reported species, respectively. Similar results

were also reported by Hussain et al. [32], where the highest SUV represents the most exploited

medicinal plants used to treat a specific ailment.

In total, 30 medicinal plant species have been recorded in our study. All belong to 20 differ-

ent families, with Poaceae and Zingiberaceae being dominant in the study area (each consist-

ing of four species) followed by Apiaceae (three species). The remaining families were

represented by one or two species (Table 2). Poaceae and Zingiberaceae were the most repre-

sentative medicinal plant families in our study. This finding might be due to the high accessi-

bility of these species in that region. This further supports that dominant plant families and

species are commonly used by local people for disease treatment [32]. Moreover, most of the

species within both families are cultivated by people of Tengger in the Ngadisari village. The

occurrence of dominant plant species and families in the study area is also related to favorable

climate and environmental conditions [33, 34]. As a result of the abundance, these species are

commonly used as a basic ingredient of Jamu—an Indonesian traditional medicine [35]. Shar-

ifi-Rad et al. [36] also described that plants from the Zingiberaceae family are a potential

source of bioactive phytochemical.

The total number of species within a given family has been calculated to obtain their FUV.

Our results showed that Aloaceae had a high FUV (0.86), followed by Acoraceae (0.80), Pipera-

ceae (0.69), and Euphorbiaceae (0.65). Other families represented low FUV (< 0.60) (Table 2).

High values of FUV might be because the plant species were cited by a large number of people

in the study area. In addition, some reports have described similar results. For example, A.

vera or locally named as crocodile’s tongues has been frequently used in some regions such as

Southern Africa [37], Asia [38], Nigeria [39], and India [40] to treat dry skin, for improving

skin integrity, and to decrease the appearance of acne, skin burn, and wrinkles.

A. calamus is also cited by other ethnobotanical studies around the world including China

[41], India [42], Nepal [43] to treat fever, diarrhea, bronchitis, tumors, skin diseases, and

Table 1. (Continued)

No Disease Categories Specified disease

name

Plant family Plant species Common name Local name Plant part

used

Mode of

preparation

5 Skeleto-muscular disorders hyperuricemia Euphorbiaceae Jatropha curcas L. Jatropha Jarak Pagar Leaves Decoction

Muscle soreness Poaceae Dendrocalamus asper(Schult. f.) Backer ex

Heyne

Dragon bamboo,

giant bamboo

Bambu betung Stem Pounded

6 Gastrointestinal disorders Diarrhea Apiaceace Coriandrum sativum L. Coriander ketumbar Stem Burned

Convolvulaceae Ipomoea paniculata Burm.

f.

Bindweed Tirem Leaves Decoction

Myrtaceae Psidium guajava L. Guava Jambu klutuk Fruit Eaten raw

Constipation Brassicaceae Brassica sp. Mustard Sawi Tengger Leaves Decoction

Worm disease Piperaceae Piper betle L. Betelvine Sirih Leaves Decoction

7 Others Motion sickness Apiaceace Foeniculum vulgare Mill. Fennel Adas Leaves Squeezed

https://doi.org/10.1371/journal.pone.0235886.t001





cough treatment [29]. Reported species from Piperaceae has also highly cited in the previous

study [11]. Finally, another high FUV in this study was obtained by Euphorbiaceae with only

one species (Jatropha curcas). The Tengger people use this Barbados nut species to treat mouth

ulcers and hyperuricemia. Our data supports other studies; for example, Abdelgadir and Sta-

den [44] reported that its latex is used for ailments such as headache, toothache, mouth ulcers,

cold, and cough. Abu Bakar et al. [45] also reported that J. curcas is potentially used to treat

hyperuricemia.

Fig 2. Species Use Value (SUV) of medicinal plants found in the Ngadisari village, Indonesia.






Fidelity level

According to Imran et al. [46], the fidelity level (FL) is useful to determine the level of species

importance in relation to a particular disease. FL shows the percentage of respondents who

mention the use of a plant species for the same main purpose. Ouedraogo et al. [21] reported

that relative frequency of citation could also be counted based on FL. This is designed to mea-

sure species importance for specific purposes. Our results showed that FL of the 30 plant spe-

cies ranged from 1.92 to 80% (Table 3). A. calamus demonstrated the highest FL for fever

(80%), followed by A. graveolens (76.92%) and A. fistulosum (71.15) for treating hypertension

and cough, respectively. Based on a previous study, plants with a high percentage of FL are

more frequently used as bio-pharmacological resources [47] and should be considered for fur-

ther conservation program [48] bioassays and phytopharmacological investigation [49, 50].

Some species have low percentage of FL (1.92%) related to various diseases (Table 3). Exam-

ples include, S. muricatum, Z. zerumbet, C. nardus, C. domestica, P. guajava, G. mangostana, P.

foetida, S. alata, K. galanga, C. sativum, Brassica sp. Low fidelity levels might also explain the

low abundance of plant species in this region. Furthermore, it might also indicate that there is

little information about the use of this medicinal plant among the people of Tengger in the

Table 2. Family Use Value (FUV) of medicinal plants found in Ngadisari village, Indonesia.

No. Plant Family FUV Number of Species Local name (Plant species)/voucher number

1 Apiaceace 0.59 3 Adas (Foeniculum vulgare Mill.) / NAD-003

Sledri (Apium graveolens L.) / NAD-005

Tumbar (Coriandrum sativum L.) / NAD-001

2 Acoraceae 0.8 1 Dringu (Acorus calamus L.) / NAD-030

3 Arecaceae 0.28 1 Kelapa (Cocos nucifera L.) / NAD-019

4 Asteraceae 0.30 1 Ganjan (Artemisia vulgaris L.) / NAD-021

5 Aloaceae 0.86 1 Lidah buaya (Aloe vera (L.) Burm. f.) / NAD-006

6 Apocynaceae 0.28 1 Bunga trompet (Mandevilla sanderi (Hemsl.) Woodson) / NAD-029

7 Brassicaceae 0.01 1 Sawi tengger (Brassica sp.) / NAD-002

8 Clusiaceae 0.01 1 Manggis (Garcinia mangostana L.) / NAD-023

9 Euphorbiaceae 0.65 1 Jarak Pagar (Jatropha curcas L.) / NAD-015

10 Liliaceae 0.41 2 Bawang prei (Allium fistulosum L.) / NAD-010

Bawang merah Tengger (Allium cepa L.) / NAD-009

11 Myrtaceae 0.32 1 Jambu (Psidium guajava L.) / NAD-014

12 Piperaceae 0.69 1 Sirih (Piper betle L.) / NAD-008

13 Poaceae 0.1 4 Serai (Cymbopogon nardus (L.) Rendle) / NAD-017

Bambu betung (Dendrocalamus asper (Schult. f.) Backer ex Heyne) / NAD-022

Tebu merah (Saccharum officinarum L.) / NAD-018

Alang-alang (Imperata cylindrica (L.) P. Beauv.) / NAD-016

14 Polypodiaceae 0.03 1 Pangotan (Microsorum buergerianum (Miq.) Ching.) / NAD-012

15 Rutaceae 0.03 1 Jeruk nipis (Citrus aurantium L.) / NAD-013

16 Rubiaceae 0.01 1 Kesimbukan (Paederia foetida L.) / NAD-024

17 Solanaceae 0.35 2 Keciplukan (Physalis angulata L.) /NAD-011

Buah melody (Solanum muricatum Ait.) / NAD-025

18 Convolvulaceae 0.28 1 Tirem (Ipomoea paniculata Burm. f.) / NAD-020

19 Zingiberaceae 0.10 4 Jahe (Zingiber officinale Rosc.) / NAD-004

Kunyit (Curcuma domestica Val.) / NAD-028

Lempuyang (Zingiber zerumbet (L.) Sm.) / NAD-007

Kencur (Kaempferia galanga L.) / NAD-026

20 Fabaceae 0.01 1 Ketepeng (Senna alata (L.) Roxb.) / NAD-027






Ngadisari village. Even though some plants possess low FL, these species should not be aban-

doned to preserve traditional knowledge of the society in treating some diseases as reported by

Chaachouay et al. [51].

Plant part use and mode of preparation

According to Hoffman and Gallaher [22], calculating the use of plant parts (Plant Part Use) is

useful to determine the dominant plant parts being used as medicinal ingredients. Plant parts

Table 3. Fidelity Level (FL) of medicinal plants in Ngadisari village, Indonesia.

Disease categories Plant Species Specified disease name Fidelity level (%)

Internal medical diseases Apium graveolens L. Hypertension 76.92

Physalis angulata L. Hypertension 38.46

Solanum muricatum Ait. Hypertension 1.92

Zingiber zerumbet (L.) Sm. Hypertension 1.92

Acorus calamus L. Fever 80

Allium cepa L. Fever 11.50

Cymbopogon nardus (L.) Rendle Fever 1.92

Saccharum officinarum L. Fever 3.84

Curcuma domestica Val. Fever 1.92

Artemisia vulgaris L. Nose bleeding 30.76

Psidium guajava L. Hemorrhoid 1.92

Physalis angulata L. Hemorrhoid 30.76

Garcinia mangostana L. Hemorrhoid 1.92

Ureno-genital and gynaecological problems Piper bettle L. Leucorrhoea 42.30

Paederia foetida L. Leucorrhoea 1.92

Dermatological diseases Cocos nucifera L. Hair problems, hair nourisment 28.84

Aloe vera (L.) Burm. f. Hair problems, hair nourisment 65.38

Foeniculum vulgare Mill. Itchy, urticaria/hives 36.53

Piper bettle L. Itchy, urticaria/hives 3.38

Microsorum buergerianum (Miq.) Ching. Itchy, urticaria/hives 3.84

Senna alata (L.) Roxb. Ringworm 1.92

Aloe vera (L.) Burm. f. Skin burn 21.15

Respiratory-nose, ear, oral/dental, throat problems Allium fistulosum L. Cough 71.15

Foeniculum vulgare Mill. Cough 42.30

Citrus aurantium L. Cough 3.84

Zingiber officinale Rosc. Cough 36.54

Kaempferia galanga L. Cough 1.92

Jatropha curcas L. Sprue, mouth ulcer 65.38

Cymbopogon nardus (L.) Rendle Asthma 1.92

Imperata cylindrica (L.) P. Beauv. Heatiness 3.84

Mandevilla sanderi (Hemsl.) Woodson Eye irritation 28.84

Skeleto-muscular disorders Jatropha curcas L. Hyperuricemia 3.38

Dendrocalamus asper (Schult. f.) Backer ex Heyne Muscle soreness 30.76

Gastro-intestinal disorders Coriandrum sativum L. Diarrhea 1.92

Ipomoea paniculata Burm. f. Diarrhea 28.84

Psidium guajava L. Diarrhea 32.69

Brassica sp. Constipation 1.92

Piper bettle L. Worm disease 23.07

Others Foeniculum vulgare Mill. Motion sickness 3.83






are capable of accumulating diverse and interesting natural compounds. They attract attention

because of their ability to act as factories, producing and offering important pharmaceutical

potential [52]. Our results showed that leaves were the most predominantly utilized plant parts

at 61.5%, while gum, stem bark and bulb represent parts that are infrequently used by people

of Ngadisari (Fig 3).

Leaves are the major plant components commonly reported to be used as herbal medicine

materials in Indonesia [8, 53] and also in other countries [54–56]. Leaves are common and

favorite parts used for medicinal treatment preparation because of easy handling and sustain-

ability [57, 58]. The latter is linked to the survival rate of medicinal plants. Removing the leaves

biomass within reasonable limits does not interfere with the plant life, compared to collecting

the stem, root, or whole plant, which may risk the plant life [59]. Moreover, many reports have

showed that leaves contain diverse plant secondary metabolites [60]. In the present study, no

data have been obtained for the use of flowers as medicinal materials. This might offer other

perspectives for further investigation. Furthermore, our data have also shown the use of more

than one plant part from the same plant species. For instance, J. curcas leaves and stem have

been used to treat hyperuricemia and mouth ulcer, respectively.

People from the Ngadisari village use many methods to prepare plant parts before using

them as herbal medicine. The decoction is considered the main mode of preparation (40.9%),

followed by pounding (15.9%) and burning (13.6%). Meanwhile, eating raw (9.1%) and smear-

ing (6.8%) contribute and of the total mode of preparation in the present study. Other miscella-

neous modes of preparations constitute the remaining 13.6% (Fig 4). Some other studies have

also mentioned the same results, where the most common method of preparation is decoction

[61–63]. Simple, easy handling and inexpensive are the major reasons why this mode of prepa-

ration is widely used by society [64]. Moreover, other reports also demonstrated that decoction

might increase the efficiency of plant extraction and therefore increase its bioactivity [65].

Some plants can be prepared without any processing. For example, leaves of A. graveolensare eaten raw to reducing hypertension symptoms and leaves of A. vulgaris are applied directly

Fig 3. Percentage of medicinal plant part use for herbal preparation in Ngadisari village, Indonesia.






by clogging into the nose to stop nosebleeds. All this local knowledge is preserved and applied

by the people of Tengger in Ngadisari village. This practice is common in other regions in

Indonesia such as in Madura and Bali [5, 66].

Conclusions

Our results highlighted the use of medicinal plants by people from the Ngadisari village, Indo-

nesia. A total of 30 medicinal plant species were recorded in the present study. They belong to

20 different families, where Poaceae and Zingiberaceae were the most representative families.

A high number of plant species were used for treating internal medical diseases, respiratory-

nose, ear, oral/dental, and throat problems. Leaves were the most popular plant part used and

decoction was the most common method of preparation. These findings indicated potential

roles of medicinal plants used in the Ngadisari village. Furthermore, our study characterized

the cultural values of the people of the Ngadisari village. The species use, family use values and

fidelity levels presented here may be used to further support plant conservation and pharmaco-

logical studies for new drug discovery. Out of all the plants we reported, approximately 26.7%

were novel medicinal plants. In addition, 71.8% of the plant uses we documented of medicinal

species were also novel. Some highly cited species recorded in our study warrant further bio-

chemical analyses to evaluate their bioactive substances. Moreover, in vitro plant tissue culture

could also be used as an alternative way to conserve medicinal plants documented in this

study. Finally, the information we obtained could enable the local communities to develop,

market, and profit from dried herbal products, which then substantially improving the collec-

tive revenue of the local society.

Acknowledgments

We would like to thank to all people from the Ngadisari village and the people of Tengger for

participating in this study and sharing all their information.

Fig 4. Mode of preparation of the medicinal plants used by the people of Tengger in Ngadisari village, Indonesia.






Author Contributions

Conceptualization: Nurul Jadid, Kristanti Indah Purwani.

Data curation: Erwin Kurniawan.

Formal analysis: Nurul Jadid, Chusnul Eka Safitri Himayani, Andriyani, Indah Prasetyowati,

Kristanti Indah Purwani, Wirdhatul Muslihatin, Dewi Hidayati, Indah Trisnawati Dwi

Tjahjaningrum.

Funding acquisition: Nurul Jadid.

Investigation: Erwin Kurniawan.

Methodology: Nurul Jadid, Erwin Kurniawan, Kristanti Indah Purwani, Wirdhatul Musliha-

tin, Dewi Hidayati, Indah Trisnawati Dwi Tjahjaningrum.

Supervision: Nurul Jadid.

Visualization: Chusnul Eka Safitri Himayani, Andriyani, Indah Prasetyowati.

Writing – original draft: Nurul Jadid, Erwin Kurniawan.

Writing – review & editing: Nurul Jadid.

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Differential Responses of Tomato (Solanum Lycopersicum Mill.) Against Toxic Heavy

Metal Contamination : A Review

Chusnul Eka Safitri Himayani, Kristanti Indah Purwani, Wirdhatul Muslihatin, Tutik

Nurhidayati, Nurul Jadid*

Departement of Biology, Faculty of Science and Data Analytics, Institut Teknologi

Sepuluh Nopember (ITS), Surabaya, Indonesia

*Corresponding author (NJ)

Email : [email protected]

mailto:[email protected]

ABSTRACT

A nutritional value contained in tomato (Solanum lycopersicum Mill.) makes the consumption

of this potential horticultural crop tends to increase each year. A lycopene-the principal

carotenoid found in tomato has been investigated for its antioxidant properties and has attracted

much attention to be developed as an anti-cancer. Hence, tomato cultivation and its quality

improvement become indispensable. However, massive horticultural crop cultivation ends up

frequently with heavy metal contamination in soils. The later causes severe problems in plant

growth and development, including in tomato. Many reports have been demonstrated that

heavy metals (Cu, Pb, Cd, Fe, Ca, Al, Hg) contamination cause a decrease in plant productivity

and might further lead to crop failure. Metallothioneins (MT) are a family of cysteine-rich

protein that is used as a biomarker for assessing plant response to heavy metals. This review

focuses on the genetic regulation governing the expression of gene encoding MT. In addition,

toxic heavy metal ions also perturb protein folding system, leading to disruption on cellular

protein homeostasis and reduction of plant cell viability. Interesting phenomena also found in

various varieties of tomato which perform different response following exposure to an excess

of heavy metals. It includes morphological and biochemical changes, suggesting that genetic

variability influences plant mechanisms against heavy metal stress.

Key words : Heavy metal, Metallothioneins, Protein Folding, Solanum lycopersicum.

Introduction

Tomato (Solanum lycopersicum L.) is an important horticultural commodity that is

frequently cultivated around the world. Tomato fruit contains vitamins and minerals that are

good for human health. In addition, major carotenoid found in its fruit (lycopene) has been

reported to be potential antioxidant and anti-cancer [1]. Therefore, fresh tomato fruit demand

increases significantly in the world market. Consequently, tomato seed production industry

become essential. Also, creating of new tomato varieties through targeted plant breeding is

necessarily needed. However, unpredicted climate change and environmental stresses such as

salinity, drought, cold, air pollution, high temperature stresses and heavy metals are still the

major obstacle in tomato cultivation. In addition, it also limits plant productivity [2]. Among

those environmental stresses, heavy metals have become main problem in this industrialization

era. Anthropogenic perturbation through industrial activities and excess use of fertilizer as well

as pesticides in horticultural cultivation increase the risk of heavy metal contamination in soils.

Heavy metals are found naturally and possess high atomic weight that greater than water.

Therefore, agricultural activities are prone to heavy metal contamination through their regular

irrigation [3]. The use of inorganic fertilizers and pesticides have been reported to contribute

to soil contamination from Copper (Cu), Lead (Pb), cadmium (Cd), iron (Fe), Aluminum (Al),

mercury (Hg) [4].

Exposure to heavy metal contamination can disrupt physiological, cellular and molecular

mechanisms in tomato plant [5]. For instance, toxic effect of heavy metals has been reported

to generate growth retardation, early senescence, and many other physiological and

biochemical disorders [6]. Inhibition of mineral distribution and enzyme activities are also

occurred because of heavy metal stress. The later include photosynthetic mechanisms including

inhibition of chlorophyll and other secondary pigments biosynthesis. Consequently, there will

be a cascade of inhibition of plant productivity [7]. Some negative growth and physiological

responses were reported in Solanaceae plants including Capsicum frutescence due to excess of

copper in soils. Growth reduction caused by copper contamination include plant height and

root reduction [8]. Also, a decrease of chlorophyll content was demonstrated as a negative

physiological response of C. frutescence to copper stress. Our previous study demonstrated

also that those responses might be due to overproduction of reactive oxygen species (ROS) that

is generated during heavy metal stress [9].

Many biological processes depend on the functionality of specific proteins. The structural

conformation of the proteins relies on the physical and chemical condition of the environment

affected by both biotic and abiotic stresses. Many reports have demonstrated that

environmental stress could disrupt de novo protein folding mechanisms ([10];[11]).

Furthermore, it also induces mis-folding of some existing proteins. Further consequences might

be the generation of endoplasmic reticulum (ER) stress which induces finally to a decrease cell

viability. Therefore, study on how cell performs protein quality check as well as heat shock

protein (HSP) as a central protein that function as protein surveillance mechanisms are

necessarily needed [12].

Mechanism of detoxifying heavy metals is vital for plant growth and development. One

of protein that play an important role during heavy metal stress is metallothionein (MT). It

includes heavy metal sequestration, maintaining protein homeostasis, and cellular protection

against oxidative stress generated by heavy metals accumulation. Metallothioneins (MT) are a

family of cysteine-rich protein that is used as a biomarker for assessing plant response to heavy

metals [13]. Although many reports have been made for elucidating plant responses to heavy

metal stress, information about how tomato response to heavy metal stress and genetic

regulation governing the expression of gene encoding MT still needs to be further explored. In

this present review, we aim to provide information of metallothionein as an important protein

regulating the detoxification of heavy metal and some physiological and molecular responses

of tomato under heavy metal stress.

Solanum lycopersicum L.

Tomato (Solanum lycopersicum L.) is one of Solanaceae plants which offer many

nutritional values. It contains vitamin A, Vitamin C, potassium, phosphorus, magnesium and

calcium [14]. Moreover, tomatoes also contain antioxidants which come from many potential

substances including terpenoids, polyphenols, flavonoids, tannins, anthocyanins, and many

other compounds [15]. Some of those substances function not only to reduce the oxidative

stress generated by reactive oxygen species (ROS), but they also modulate protection against

lifestyle disorders such as diabetes, obesity, cardiovascular and cancer [16].

Tomato has been thought to be originated from Peru and is probably domesticated in

Mexico [17]. This horticultural crop then has been spread around the world, including in

Indonesia. Its worldwide production reach more than 182 million tons (Figure 1) [18].

According to [19], more than 7500 tomato landraces and varieties are bred in the world. Some

of them can tolerate certain environmental stress and can be planted in the lowlands or in

highlands. Generally, tomato that are suitable to be cultivated in low land are drought or heat

resistance varieties and pathogenic resistance varieties. Some Indonesian tomato varieties

include var. Intan, Ratna, Berlian, Mutiara, Mirah, Opal, Emerald, Rempai, Rose and many

other varieties [20].

Figure 1. Production/yield quantities of tomatoes in the world [18].

Many genetic diversity studies have been conducted since tomato varieties are

continually developed. It includes development of DNA-based markers and phylogenetic

reconstruction among tomato varieties. Those are restriction fragment length polymorphism

(RFLP), random amplified polymorphic DNA (RAPD) [21], amplified fragment length

polymorphism (AFLP) [22]. Another molecular marker such as simple sequence repeat (SSR)

has been also used to assess the diversity of tomatos, even though previous report has

demonstrated low polymorphism shown using SSR [23]. Sequence-related amplified

polymorphism (SRAP) [24], single nucleotide polymorphism (SNP) [25] have been also

reported as potential tools to analyze the genetic diversity of tomatoes.

Morpho-physiological Responses of Tomato against Heavy Metal Stress

Unfavorable environmental condition has forced plants to develop fascinating

adaptation mechanism. It involves biochemical and physiological processes. Activation of

antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione and

ascorbate peroxidase (APX) play important role during oxidative stress, which is usually

generated by environmental stress [26]. Other antioxidant metabolites including ascorbic acid,

carotenoids, tocopherol, proline, alkaloids and flavonoids have been also reported to be highly

produced during environmental stress [27]. Many plant proteomic studies have demonstrated

diverse plant protein function during plant response to abiotic stress including heavy metal

stress. Those proteins control metal detoxification [28]. In addition, plant genomic studies also

showed that many genes are involved in the regulation of plant under heavy metal stress [29].

Also, protein-protein interaction as well as protein folding process (chaperones) are vital for

regulating plant adaptation against heavy metal stress [30].

Metallothioneins (MT)

One of the most reactive plant proteins during heavy metal stress is metallothioneins (MTs).

These proteins are considered as intracellular cystein-rich proteins (30% of the total amino acid

content) that are capable of binding metal. They are synthesized across organisms including

prokaryote (bacteria) and eukaryote (plants, fungi, and animals) [27]. Due to their important

roles, these proteins are potential to be developed as molecular markers [13]. Interestingly,

plant MTs function not only for maintaining cellular homeostasis and metal detoxification but

MTs are also play important role in plant development including fruit ripening [31], root

growth development [32] and free radicals scavenging [33].

Plant MTs have been characterized as low molecular weight proteins. However, the plant

MTs are still higher in their molecular mass, compared to those in animal. This is due to their

longer amino acid sequences. Commonly, animal MT is around 6 KDa, while plant MTs

ranging from 6.0 to 7.6 KDa. Based on its structure, MT possess two subunits: stable α domain

(C terminal) and a reactive β-domain (N terminal) (Figure 2). Basic spatial structure of MT is

in the form of dumbbell with two separate domains. According to [34], MTs can bind to various

metal ions. However, MTs show high affinity to Cu, Cd and Zn, respectively. This

characteristic is mainly due to the different functional roles of the two subunits. C-terminal of

the protein (α domain) strongly binds to an excess of toxic metal ions [35].

Figure 2. Spatial structure of metallothionein. (A) General structure of MT: shaped like a

dumbbell with two separate globular domains α and β; (B) The domain structure (Cd 4) α of

the MT2 rat shows an example of the Me (II) -Cys tetrahedral unit formed by MT (adapted

from [36]).

Based on the arrangement of its Cysteine residues, plant MTs have been grouped into four

types : MT type 1, MT type 2, MT type 3, and MT type 4. MT type 1 is composed of six Cys-

motifs X-Cys (-X- is an amino acid other than cysteine). These motifs are present in both

terminals. MT type 2 has two Cys-rich domains which are separated by long spacer. Type 3

MT only consists of four Cys amino acids at the N-terminal end. The first three Cys are in the

form of Cys-Gly-Asn-Cys-Asp-Cys motif. Whereas the fourth Cys forms its own motif namely

Gln-Cys-X-Lys-Lys-Gly. Finally, the MT type 4 has three Cys-rich domains. Each Cys-rich

domain has 5 to 6 conserved Cys residues [37].

MTs genes mapping of tomato plants demonstrated that the four types of MTs possess

different characteristic patterns and are expressed in various tissue in tomato plants. Specific

cDNAs encoding tomato metallothionein-like proteins have been characterized. These include

LEMT1, LEMT2, LEMT3 and LEMT4. [38] revealed that LEMT1, LEMT3 and LEMT4

belong to the MTs type two. Different transcriptional expression of the MTs genes in tomato

was also observed by [39]. S. lycopersicum under Cd stress exhibit distinct molecular

responses. MT1 and MT2 genes were upregulated during the Cd stress in root, fruit and leaves

of tomatoes. Meanwhile, MT3 and MT3 expression changed in term of type of tissue.

Moreover, Cd level also increased in the root, fruit and leaves of the plant. Other minerals such

as Mg, Ca and fe were also accumulated on the leaves and fruits. Thus, Cd could synergistically

or antagonistically affect minerals uptake in plants [39].

Many studies have been conducted to improve plant adaptation ability against heavy metal

stress using MTs genes. Overexpression of Salicornia brachiata Mt2 (SbMt2) gene has been

reported to regulate free radicals scavenging and increase plant tolerance to heavy metal stress

[40]. Moreover, transgenic Arabidopsis overexpressing OsMT2c has also reported to exhibit

enhance Cu tolerance and ability to scavenge reactive oxygen species (ROS) [41]. Recently,

[42] has also showed that overexpressing OsMT-3a in Arabidopsis is not only increasing plant

tolerance to heavy metal stress (CdCl2), but also enhance the ability to tolerate an excess of

salinity and drought stress. Finally, palm date transgenic MT2 has also been reported to exhibit

higher production of enzymatic antioxidant (SOD) and chlorophyll content during salinity and

oxidative stress [43]. The overall studies demonstrate the potential uses of metallothionein

genes to improve plant tolerance to abiotic stress and thus for increasing plant productivity.

Protein folding and physiological process of plants during heavy metal stress

Proteins are important macromolecules that play important roles in a plethora of

biological processes. Proteins function in plant signaling, as catalysts in many biological

reactions, intra- and inter-cellular movement of nutrients, membrane fusion and protectors [44].

However, their specific function is basically influenced by their native structure obtained

during the protein synthesis. In addition, protein conformation depends on the physico-

chemical conditions of the environment. Unstable environmental conditions caused by extreme

temperatures, oxidative molecules and heavy metal can interfere the protein folding process.

Consequently, it could induce frequent errors in the protein production [45].

Plants response to abiotic stresses including heavy metals by triggering the expression

of protein-coding genes involved in the stress response. Heavy metal ions greatly influence

cellular protein homeostasis by interfering protein folding and causes consequently to a

decrease of cell viability [46]. Protein folding occurs in the endoplasmic reticulum (ER) and

therefore, ER possesses folding machinery tools called molecular chaperones. This includes

binding protein (BiP), calnexin (CNX) and calreticulins (CRT). This machinery function to

avoid aggregation of the denatured protein. Othe machinery involved during protein folding is

protein disulfide isomerase (PDI). This enzyme is involved in the formation of disulfide bridges

between Cys of the proteins [47].

Environmental stress, including heavy metal that received by plants could generate

protein misfolding. Several studies have shown that heavy metals stress inhibit the re-

establishment of chemically denatured proteins in vitro, and inhibit protein folding [48].

Consequently, unfolded proteins are accumulated in the ER. An excess of unfolded protein in

the ER induce subsequently the ER stress. This type of stress then transmit the unfolded protein

response (UPR) signal. UPR signal has three objectives (1) to restore the function of cell by

stopping the production of secreted membrane proteins, (2) removal of unfolded proteins, and

activation of signaling pathways that lead to increased companion molecules involved in

protein folding, (3) to tackle progressive disorders by conducting programmed cell death (PCD)

[12].

UPR is a sensitive cellular system that monitors the loading capacity of ER. The

unfolded protein accumulated in the ER results in cellular communication between ER and the

nucleus leading to transcriptional activation [49]. Transcriptional activation of UPR will

increase the production of molecular chaperon proteins [50]. Some proteins that cannot be fixed

would undergo protein degradation to maintain cellular homeostasis. The later occurs via the

Ubiquitin proteasome system (UPS) and autophagosomes [12]. UPS is a multi-step enzymatic

cascade to induce the degradation of unfolded proteins at specific times [51]. While

autophagosomes are double membrane vesicles structure that is involved in the autophagy

process. Autophagy is a process of biological self-destruction carried out by eukaryotic cells

to maintain cellular homeostasis by converting damaged proteins or organelles into vacuoles

during the developmental transition under stressful conditions [52]. After induction of the

autophagy pathway, the cytoplasmic component designated for degradation is surrounded by a

double membrane structure (Autophagosome) (Figure 3).

Figure 3. Schematic diagram illustrating the main pathway and protein fold plan and

modification in the endoplasmic reticulum (RE). New synthesized proteins are translated into

the ER, proteins are folded in a 3D structure. the protein is transported to the golgi body,

followed by the sending of the protein to its destination according to its function. Exposure to

plants exposed to oxidative stress results in excessive ROS and stimulates protein for wrong

folds. Incorrect protein folds are detected by a quality control system that will stimulate (UPR).

The wrong folded protein is then removed through the endoplasmic reticulum (RE) and

degraded through (ERDA). This process begins with ubiquitin being degraded in the cytoplasm

by the proteasome system (UPS) or experiencing autophagy. Adopted from [53] in [46] with

modifications.

Different tomato varieties exhibited distinct responses to heavy metal ions

Tomatoes are one of the main vegetable crops throughout the world, contributing vitamin

A and vitamin C bioresources. In addition, tomato plants are also considered as leading model

crops for genetic studies in plants [54]. Therefore, extensive studies of genetic diversity,

molecular response and physiological studies have been conducted. For instance, [55] have

analyzed responses of about hundred tomato genotypes under cadmium stress. It has been

stated that they exhibited different responses. Some genotypes demonstrated a minimum level

of Cd. Heavy metal has been reported to accumulated first in the shoot, fruit, leaves and root.

However, in the susceptible tomato genotype, the Cd was accumulated first in the fruit, shoot,

leaves and root.

Other study conducted by [56] showed that S. lycopersicum that was planted in the

contaminated soils demonstrated negative effect on the fruit characteristics, lycopene, ascorbic

acid, and carbohydrate content. Moreover, tomato fruits have been reported to accumulate large

quantity of phenols and flavonoids. [57] also reported the negative consequences of heavy

metal stress in tomato productivity. A reduction of fruit dry weight was observed following an

increase of CdCl2 concentration in the soils. Whereas no specific results were observed in term

of chlorophyll content. Some tomato plants exhibited an increase of chlorophyll content under

Cd stress, while others showed a decrease of chlorophyll content.

Chlorosis and necrotic spot are also occurred when tomato is grown under 10 M and 100

M of Cd, respectively. Rootbrowning is another symptom that might be occurred because of

the Cd stress. Meanwhile, biochemical studies revealed an increase of phosphoenolpyruvate

carboxylase accumulation in the tomato root grown in Cd-contaminated hydroponic system.

Accumulation of citrate synthase, isocitrate dehydrogenase and malate dehydrogenase were

also found in the tomato leaves [58]. The overall studies demonstrated that heavy metal stress

might perturb photosynthetic rate and pigment, enzymatic reactions, and morphological

alterations of tomato plants.

Effect of heavy metal Pb on gene expression in Solanum lycopersicum

Heavy metal Pb is a destructive heavy metal that occurs naturally in the earth's crust [59]

and originates from a variety of anthropogenic activities such as smelting ore, the battery

industry, paint, exhaust, and fossil fuel combustion [4]. The higher the concentration of Pb

heavy metal available in the soil, the higher the absorption of Pb in plants. The accumulation

of high amounts of Pb in plants will cause changes in chloroplast structure, imbalance in

nutrient absorption, and induce reactive oxygen ordering (ROS) which will inhibit enzyme

activity, reduce protein and affect gene expression [60].

In stressful conditions, plants will respond to signals from the environment to protect and

reduce the harmful effects of stress. One of the plant protection responses is the molecular

response. The molecular response can be identified through the expression of genes that appear

when the plant is in a state of stress. Gene expression that occurs in plants will show the defense

system and plant metabolism [61].

Gene expression that occurs when plants are stressed by heavy metals is shown by up-

regulation or down-regulation of genes sensitive to heavy metals. Up-regulation and down-

regulation of genes are influenced by the function of these genes. Data on several gene

expressions sensitive to heavy metals can be seen in (Table 4.4).

No

Nama Gen

Fungsi Gen

Respon

Referensi Up

Regulasi

Down

Regulasi

1. LEMT-1 Encodes Metallothionein

Biosynthesis type 1

√

-

[39]


Biosinthesis type 2

√

-

[39]


Biosinthesis type 3

√

-

[39]


Biosinthesis type 4

√

-

[39]

5. LEHsp 90 -1 Encodes HSP-90 (Heat

shock protein/Chaperon)

Biosinthesis

√

-

[62]

6. NCED 2/3 Encodes ABA (Abscisic

Acid) Biosinthesis

√

-

[63]

7. PIN1 Auxin hormone transport

mechanism

-

√

[63]

8. EIN 2 Ethylene hormone signalin

mechanism

√

-

[63]

9. P5CS1 Encodes Proline

Biosinthesis

√

-

[64]

10. GSH Encodes GR (Gluthation

reduxtase) Biosinthesis

√

-

[65]

Table 4.4 shows some of the gene expressions that occurred in Solanum Lycopersicum

by giving heavy metal stress Pb. The gene expression shown in table 4.4 can be grouped into 3

groups based on their function, namely:

1. Genes play a role in protein folding

Genes that play a role in protein folding are the LEMT-1, LEMT-2, LEMT-3, LEMT-4,

and LEHsp90-1 genes.

a. Genes LEMT-1, LEMT-2, LEMT-3, LEMT-4.

The LEMT-1, LEMT-2, LEMT-3, LEMT-4 genes are genes that encode the biosynthesis

of Metallothionein type 1, type 2, type 3, and type 4 (Kisa et al., 2016). Metallothionein is a

polypeptide that has cysteine bonds (cys) and has sulfide residues (-SH). The different types of

metallothionein are structurally and their location is different in plant parts [66]. The structural

pattern (cys) and residual sulfide (-SH) possessed by metallothioneins are able to bind toxic

heavy metals, heavy metal detoxification, molecular markers maintain cell homeostasis,

development of root growth and free radical scavengers ([67]; [13]; [32]; [33]).

In high heavy metal stress, the LEMT gene will be up-regulated and MT (Metallothionein) will

be available in large quantities. Large amounts of MT will bind toxic heavy metals to maintain

cell homeostasis [67].

b. LEHsp90-1 gene

The LEHsp90-1 gene is a gene that codes for Chaperon biosynthesis [62]. chaperons are

proteins that assist in non-covalent folding and unfolding as well as the attachment and release

of proteins with other macromolecular structures. Chaperons function especially if there are

protein folding problems [68].

Under stress conditions, misfolded and aggregated proteins will cause fungal disorders of

the Endoplasmic Reticulum (ER). The impaired function of the ER is called the RE pressure

[69]. The wrongly folded protein will give a signal to the RE and will make Chaperon up-

regulate to correct the misfolded protein.

2. Genes play a role in Hormone Biosynthesis

a. Gen NCED 2/3

The NCED 2/3 gene is a gene that encodes the biosynthesis of the hormone ABA (Abscisic

Acid) (Bücker-Neto et al., 2017). In stressful conditions, the NCED2 / 3 gene will be

upregulated and will increase the endogenous ABA of a plant. Endogenous ABA undergoes

upregulation, then ABA will perform signal transduction on PYL / PYR / RCAR and plants

will respond to ABA [63]. ABA functions in inhibiting heavy metals from gripping plants,

preventing a decrease in water potential, and contributing to the adaptation of plants to stressful

conditions [63].

b. PIN1 gene

The PIN1 gene is a gene involved in the transport of auxin hormones [63]. In stress

conditions, the PIN1 gene will experience down-regulation, because heavy metals will induce

the presence of nitric oxide (NO) which will inhibit auxin transport and result in inhibition of

root growth [70]. Heavy metals will induce NO (Nitric oxide) accumulation which will

suppress auxin transport and under stress, the auxin heavy metal will modulate the activity of

catalase, peroxidase, and reduce the concentration of hydrogen peroxide [63].

c. EIN 2 gen

The EIN2 gene is a gene involved in the signaling mechanism of Ethylene Hormone [63].

In stressful conditions, the EIN2 gene will be upregulated. The heavy metal Pb that is available

in the soil will be absorbed by plants then it will regulate the height of the EIN2 gene and there

will be a synthesis of ethylene hormone. The ethylene hormone is synthesized from methionine

which will be converted into SAM (S-adenosylmethionine) by SAM synthase. SAM will form

ACC by ACC synthase and will form MTA. In a high O2 state, ACC is degraded by ACC

oxidase and will form ethylene [63].

3. Genes play a role in antioxidant metabolites

a. P5CS1 gene

The P5CS1 gene is a gene that encodes the biosynthesis of Proline [64]. In stressful

conditions, the P5CS1 gene will be upregulated to be synthesized into proline. Proline functions

as an osmolyte, a secondary antioxidant, and degradation of proline which is used for energy

reserves in plant growth after conditions are stressful and binds heavy metals ([72]; [73]).

b. GSH gene

The GSH gene is a gene that codes for the biosynthesis of GR (Glutathione reductase) [65].

Glutathione reductase is an important (non-enzymatic) thiol compound in cells. In stressful

conditions, the GSH gene will undergo upregulation which is much rooted and will detoxify

heavy metals [62].

Acknowledgement

This work was financially supported by the Ministry of Research and Technology/the National

Agency for Research and Innovation (BRIN), the Republic of Indonesia.

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https://scholar.google.com/scholar_lookup?journal=Plant+Cell+Physiol&title=Differential+roles+of+PIN1+and+PIN2+in+root+meristem+maintenance+under+low-B+conditions+in+Arabidopsis+thaliana&author=K+Li&author=T+Kamiya&author=T+Fujiwara&volume=56&publication_year=2015&pages=1205-1214&pmid=25814435&

http://dx.doi.org/10.1016/

https://www.its.ac.id/biologi H Building, ITS Sukolilo Campus

Jl. Raya ITS, Sukolilo, Surabaya – 60111

The 5th IBOC 2020 Biology Department Institut Teknologi Sepuluh Nopember

Surabaya, September 24th 2020

To vf

Dear Author(s)

On behalf of the organizing committee of 5th IBOC (International Biology Conference) 2020, we are

very pleased to inform you that your abstract of the paper entitled:

is accepted for oral presentation in 5th IBOC 2020. Concerning to this status, we would like to invite

you to present your full research paper on the conference at October 17th, 2020 via online at the

Zoom meeting and livestreaming YouTube.

Selected papers will be published in IOP Conference Series Earth and Environmental Science

(Scopus indexed). The detail information about the schedule will be informed.

Finally, we would like to take this opportunity to thank you for your interest in participating and

presenting your research works at the 5th IBOC 2020. We are looking forward to meet you at the

conference day. Meantime, you may also visit our official website http://iboc.its.ac.id/ for any

update information.

Cordially yours, Farid Kamal Muzaki, M.Si. Chairman of the 5th IBOC 2020

Dr. Nurul Jadid, M.Sc.

Morpho-physiological Responses of Local Tomato Varieties (Solanum lycopersicum L.) under Lead Stress Condition

http://iboc.its.ac.id/

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