Salt Stress Responses of an Alanine Aminotransferase Knock-out Mutant of Rice (Oryza sativa L.)

국문 초록 (Abstract)

T-DNA가 표지된 집단에서 AlaAT 유전자가 깨어진 돌연변이체(alaat)를 분리하고, AlaAt1 특이 프라이머를 이용하여 유전자형을 결정하였다. Alaat의 표현형은 대조구와 비교해서 생장의 감소를 보였고, 종자 역시 작고 생산성의 감소를 보였다. 돌연변이체의 AlaAT 활성은 거의 검출되지 않았다. 고염 스트레스 하에서 alaat의 반응을 엽록소 형광과 항산화 효소들의 활성 및 RT-PCR을 이용하여 대조구와 비교하였다. 고염, 건조 및 저온과 같은 모든 비생물적 스트레스에 대한 Fv/Fm은 대조구와 alaat 둘 다 감소를 보였으며, 비생물적 스트레스에 대한 엽록소 형광은 거의 유사하였다. 항산화 효소인 peroxidase (POX)의 활성은 고염 스트레스에 의해 대조구는 증가하나 alaat에서는 오히려 감소하였다. RT-PCR에 의한 cAPX, POX 및 AlaAT mRNA의 수준을 분석한 결과, 효소 활성과 마찬가지로 AlaAt mRNA는 alaat에서 나타나지 않았고, POX2 mRNA는 대조구는 약간의 증가를 보이나 alaat는 거의 검출할 수 없었다. cAPX mRNA는 대조구와 alaat 모두 고염 스트레스에 의해 크게 증가하였다. 이 같은 결과는 AlaAT 유전자 기능의 상실은 염 스트레스 하에서 벼 식물의 생장에 대해 광합성능 보다는 항산화 효소, 특히 POX 활성 및 합성을 변화시킬 수 있음을 제안한다.

다국어 초록 (Multilingual Abstract)

An AlaAT knock-out mutant (alaat) of rice (Oryza sativa L.) was isolated from T-DNA tagging lines and the genotypes of its progeny were determined with AlaAT1-specific primers. The alaat phenotypes showed decreased growth and grain yield when compared with control plants. The activity of AlaAT1 in the mutant plants was practically undetectable. The responses of alaat plants to growth under salt stress were compared with those of control plants by measuring chlorophyll fluorescence and the activities and mRNA expression of antioxidant enzymes. All abiotic stresses tested (salt, drought, and chilling) caused a similar decrease in chlorophyll fluorescence in both alaat and wild type plants. The activity of peroxidase (POX), an antioxidant enzyme, decreased following salt treatment of alaat plants, while control plant showed an increased activity. The mRNA levels for cAPX (cytosolic ascorbate peroxidase), POX2, and AlaAT were determined by RT-PCR following salt stress. No AlaAT1 mRNA was detected in alaat plants. The POX2 mRNA showed a slightly increased level in the wild type but was not detected in alaat plants, in agreement with the activity assays. The levels of cAPX mRNA were greatly increased in both the wild type and alaat plants. The salt stress effects on rice plant growth are therefore proposed to reflect a loss of function of AlaAT, which alters the activity and synthesis of antioxidant enzymes (especially peroxidases), rather than a direct effect on photosynthesis.

목차 (Table of Contents)

• 서론
• 재료 및 방법
• 결과 및 고찰
• 감사의 글
• References
• 초록

참고문헌 (Reference)

1 Lee, C. S., "Trapping and characterization of cold-responsive genes from T-DNA tagging lines in rice" 166 : 69-79, 2004

2 Wilson, D. G., "Transaminase reactions in plants" 208 : 863-874, 1954

3 Genty, B., "The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence" 990 : 87-92, 1989

4 Lee. D. H., "The inductive responses of the antioxidant enzymes by salt stress in the rice(Oryza sativa L. )" 158 : 737-745, 2001

5 Jeong, D. H., "T-DNA insertional mutagenesis for activation tagging in rice" 130 : 1636-1644, 2002

6 Biekmann, S., "Subcellular distribution, multiple forms and development of glutamate-pyruvate(glyoxylate)aminotransferase in plant tissues" 721 : 268-279, 1982

7 Horton, P., "Studies on the induction of chlorophyll fuorescence in isolated barley protoplasts, Ⅳ, Resolution of non-photochemical quenching" 932 : 107-1153, 1988

8 Menezes-Benavente, L., "Salt stress altered expression of genes encording antioxidant enzymes in seedlings of a Brazilian indica rice(Oryza sativa L. )" 166 : 323-331, 2004

9 Gueta-Dahan, Y., "Salt and oxidative stress : similar and specific responses and their relation to salt tolerance in Citrus" 203 : 460-469, 1997

10 Fukao, Y., "Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana" 43 : 689-696, 2002

11 Lee, C. B., "Non-photochemical quenching of chlorophyll fluorescence in the green alga Dunaliella" 24 : 167-173, 1990

12 Son, D., "Nitrogen dependent regulation of the gene for alanine aminotransferase which is involved in the C4 pathway of Paniculum miliaceum" 33 : 507-509, 1992

13 Subbaiah, C., "Molecular and cellular adaptations of maize to flooding stress" 90 : 119-127, 2003

14 Bergmeyer, H. G., "Methods of enzymatic analysis" Academic Press 2246-2248, 1974

15 Good, A. G., "Long-term anaerobic metabolism in root tissue(metabolic products of pyruvate metabolism)" 101 : 1163-1168, 1993

16 Dumitru, I. F., "L-Alanine : 2-oxoglutarate-aminotransferase chromatographic purification and crystallization of the enzyme from seeds of Glycine hispida var Cheepeura" 7 : 31-44, 1970

17 Igarashi, D., "Identification of photorespiratory glutamate : glyoxylate aminotransferase(GGAT)gene in Arabidopsis" 33 : 975-987, 2003

18 Rocha, M., "Glycolysis and the tricarboxylic acid cycle are linked by alanine aminotransferase during hypoxia induced by waterlogging of Lotus japonicus" 152 : 1501-1513, 2010

19 Shrawat, M., "Genetic engineering of improved nitrogen use efficiency in rice by the tissue-specific expression of alanine aminotransferase" 6 : 722-732, 2008

20 Han, J. J., "Flower-preferential poly(A)binding(PAB)protein gene from rice" 165 : 103-112, 2003

21 Kendziorek, M., "Differential regulation of alanine aminotransferase homologues by abiotic stresses in wheat(Triticum aestivum L. )seedlings" 31 : 1105-1117, 2012

22 Choi, H. I., "Development of stress-tolerant crop plants" 4 : 53-58, 2002

23 Chen, B. J., "Cloning and characterization of a drought-inducible MYB gene from Boea crassifolia" 168 : 493-500, 2005

24 Choi, S. M., "Chloroplast Cu/Zn-superoxide dismutase is a highly sensitive site in cucumber leaves chilled in the light" 216 : 315-324, 2002

25 Jung, K. H., "Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system" 44 : 463-472, 2003

26 Asada, K., "Ascorbate peroxidase-a hydrogen peroxide-scavenging enzyme in plants" 85 : 235-241, 1992

27 Rocha, M., "Analysis of alanine aminotransferase in various organs of soybean(Glycine max)and in dependence of different nitrogen fertilisers during hypoxic stress" 39 : 1043-1053, 2010

28 Good, A. G., "Anaerobic induction of alanine aminotransferase in barley root tissue" 90 : 1305-1309, 1989

29 Sousa, C. A. F., "Alanine metabolism and alanine aminotransferase activity in soybean(Glycine max)during hypoxia of the root system and subsequent return to normoxia" 50 : 1-8, 2003

30 Edwards, M. R., "Alanine is a major end product of metabolism by Giardia lamblia : a proton nuclear magnetic resonance study" 37 : 19-26, 1989

31 Liepman, A. H., "Alanine aminotransferase homologs catalyze the glutamate : glyoxylate aminotransferase reaction in peroxisomes of Arabidopsis" 131 : 215-227, 2003

32 Miyashita, Y., "Alanine aminotransferase catalyses the breakdown of alanine after hypoxia in Arabidopsis thaliana" 49 : 1108-1121, 2007

33 Chen, D. H., "A rapid DNA minipreparation method suitable for AFLP and other PCR applications" 17 : 53-57, 1999