analisis polimorfisme menggunakan marka rapd
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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.
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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
* nuruljadid@bio.its.ac.id
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
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
PLOS ONE | https://doi.org/10.1371/journal.pone.0235886 July 13, 2020 2 / 16
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
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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
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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].
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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)
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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
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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.
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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
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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
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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.
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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.
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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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PLOS ONE | https://doi.org/10.1371/journal.pone.0235886 July 13, 2020 16 / 16
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 : nuruljadid@bio.its.ac.id
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]
2. LEMT-2 Encodes Metallothionein
Biosinthesis type 2
√
-
[39]
3. LEMT-3 Encodes Metallothionein
Biosinthesis type 3
√
-
[39]
4. LEMT-4 Encodes Metallothionein
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://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
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