Acetolactate synthase activity and chlorsulfuron sensitivity of

gamma-irradiated lentil (Lens culinaris Medik.) cultivars

H. I. MALKAWI, N. A. AL-QURAAN AND W. M. OWAI SDepartment of Biological Sciences, Yarmouk University, Irbid, Jordan

Department of Biotechnology and Genetic Engineering, Jordan University of Science and Technology,

Irbid, Jordan

Abdul Rosyid AMPresented by:

INTRODUCTIONChlorsulfuron [2-chloro-N-[(4-methoxy-6-methyl-

1.3.5-triazin-2-yL) amino carbonyl benzene sulfonamide] compound (trade name : Glean) (Ray 1986) belongs to the sulfonylurea class of herbicides and has been used widely in weed control (Ray 1984)

Sulfonylurea herbicides inhibit the synthesis of the three essential amino acids : isoleucine, leucine and valine (Umbarger 1978), by inhibiting the activity of the first enzyme acetolactate synthase (ALS) or acetohydroxy acid synthase (AHAS) in the synthetic pathway of these branched chain amino acids (Ray 1984)

Sensitivity of nontolerant crops is like wise due to the lack of metabolic detoxification of the herbicide in plants. Many farm crops such as barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), sugar beet (Beta vulgaris L.), corn (Zea mays L.) and lentil (Lens culinaris Medik.)

Resistance to ALS-inhibiting herbicides has been developed for many crop plants (Kuch & Bright 1981 ; Newhouse et al. 1992 ; Joel et al. 1995). Such resistance has been introduced by somatic cell selec- tion (Anderson & Georgeson 1989), mutation breed- ing, genetic engineering and interspecific hybridization (Joel et al. 1995).

MATERIALS AND METHODS Plant material The two lentil cultivars used in this study were grown

in Jordan and were provided by the National Center for Agricultural Research and Technology Transfer (NCARTT)

irradiation material Gamma-irradiation was carried out at room

temperature in a cobalt-60 gamma cell (Ministry of Energy and Mineral Resources, Amman-Jordan) of 5000 C activity and at a dose rate of 57.511 Gray/min.

Irradiation prosedureSeeds (1000 seeds/treatment) were sub- jected to gamma-irradiation with the following doses (4 treatments) : 1. 0 Gray (for 0 min), 2. 90 Gray (for 1.16 min), 3. 100 Gray (for 1.27 min) and 4. 110 Gray (for 1.37 min).

The treated seeds M1 (90, 100 and 110 Gray) and the control non-treated seeds (at 0 Gray) were grown in the field at Marrow Agricultural Station, Irbid, Jordan in a randomized complete block design with two replications for each treatment for each cultivar.

Plant treatment with herbicidewhen the plants of the treated seeds (M1) reached about 10 cm in height, chlorsulfuron (Glean,98 %, DPX, W4189-Dupont, USA) was applied at 3 g a.i./ha to all treatments using a CO2-pressurized sprayer of 5-litre capacity. Surfactant (Tween-20)

RESULTSEffect of gamma-irradiation treatment(taken after47 days of seed planting) The germination percentge of Jordan 1 was 98 % at 0 Gray, 6.2 % at 90 Gray, 5.7 % at 100 Gray and 3.4 % at 110 Gray while the percentage of germination of Jordan 2 was 95 % at 0 Gray, 5.1 % at 90 Gray, 4.3 % at 100 Gray and 1.7% at 110 Gray. Also the average plant height (cm) after complete germination was reduced as the radiation dose increased, being for Jordan 1 : 21 cm at 0 Gray, 12.5 cm at 90 Gray, 11.8 cm at 100 Gray and 10 cm at 110 Gray. For Jordan 2, the average plant height ranged from 18 cm at 0 Gray, 11.8 cm at 90 Gray, 9.5 cm at 100 Gray and 9 cm at 110 Gray

Response of lentil cultivars to chlorsulfuron

Chlorsulfuron herbicide was spread over the green, healthy and mature plants (after 7 weeks of germi- nation). In both lentil cultivars, the colour of the sensitive plants’ leaves changed from green to yellow to brown after the first week of herbicide application and died after the second week of herbicide application when compared with the healthy green tolerant plants (M1) (data not shown).

Response of lentil cultivars to chlorsulfuron

Response of lentil cultivars to chlorsulfuron

Response of lentil cultivars to chlorsulfuron

Response of lentil cultivars to chlorsulfuron

Result of Purification of ALS enzyme from lentil plants

CONCLUSION Especially at the 90 Gray treatment, The

decrease in sensitivity levels of the ALS enzyme to inhibition by chlorsulfuron herbicide might be explained by the inheritance of an alteration of the gene(s) coding for the enzyme, that could lead to overproduction of the ALS in the plants, via the increase of the expression level of the ALS gene(s),

Biology dept. Semarang State UniversitySemarang, 22 Mei 2014

evaluasiALS (asetolaktat sintase) merupakan enzim yang terbentuk saat awal pembentukan rantai cabang asam amino (valin, isolisin dan lisin)Chlorosuforon dapat: Menghambat sintesis DNA tanaman akibat produksi

asam amino terganggu Berfungsi baik pada tanaman rumput maupun dikotil Merupakan herbisida paling aman (ALS tidak terdapat

pada sel hewan) Contoh herbisida: sulfonylureas (SUs), imidazolinones

(IMIs), triazolopyrimidines (TPs), dll

Kerja chlorosurforonmenghambat kerja dari enzim acetolactate synthase (ALS) dan acetohydroxy synthase (AHAS) dengan menghambat perubahan dari α ketoglutarate menjadi 2-acetohydroxybutyrate dan piruvat menjadi 2-acetolactate sehingga mengakibatkan rantai cabang-cabang asam amino valine, leucine, dan isoleucine tidak dihasilkan. Tanpa adanya asam amino yang penting ini, maka protein tidak dapat terbentuk dan tanaman mengalami kematian (Ross and Childs, 2010).

Tanaman lentil (Lens

culinaris Medik.)

radiasi

tidak

Merubah (mutasi) susuanan DNA

DNA tetap

Menambahkan

chlorsulfuron

Menambahkan

chlorsulfuron

Enzim acetolactate synthase terhambat

drastis

Enzim acetolactate

synthase masih dapat

disintesis

lentil (Lens culinaris

Medik.) rentan terhadap

chlorosurforon

lentil (Lens culinaris

Medik.) toleran dari

chlorosurforon

Contoh penggunaan enzim acetolactate shytaseBiosintesis leusin melibatkan 8 langkah konversi, yaitu

konversi piruvat menjadi a- acetolactate, isomer a-acetolactate, a-ß- dihydroxyisovalerate, a-ketoisovalerat, aisopropylmalate, ß-isopropylmalate, a-ketoisocaproate,

dan akhrinya menjadi leucine. Proses biosintesis ini dikatalisis oleh enzim acetolactate synthase,

acetohydroxyacid isomeroreductase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase,

aisopropylmalate synthase, isopropylmalate isomerase, dihydrogenase, dan leucine

aminotransferase secara berurutan

Biosintesis leusin