Caffeine triggers calcium signaling and confers resistance against a broad-spectrum of biotic/abiotic stresses in rice

국문 초록 (Abstract)

작물은 수확량을 심각하게 감소시키는 생물학적/비 생물학적 스트레스를 비롯한 환경 요인에 지속적으로 노출된다. 이러한 스트레스에 대처하기 위해 화학 농약이 일반적으로 사용되었지만, 과도한 적용으로 인하여 종종 환경 문제가 발생한다. 따라서 기존의 방법보다 안전하고 효과적인대체 방법을 찾는 것이 시급하다. 여기에서 나는 카페인의 형질 전환접근법을 통하여 벼의 기본 면역을 높인다는 것을 보고한다. 카페인 (1,3,7-trimethylxabthine)은 퓨린 알카로이드 계열의 전형적인 2 차 대사산물중의 하나이며 커피, 카카오, 차와 같은 몇 가지 식물에서 합성된다. 카페인은 병원균과 초식동물에 독성을 일으켜 숙주 식물을 보호하는 것으로 알려져 있다. 카페인은 purine catabolism pathway에서 xanthosine 을 전구체로 사용하여 합성된다. Coffea arabica 에서 동정 된3 개의 N-메틸 전이 효소 유전자를 도입하여 카페인 생성벼(CPR)을만들었다. CPR 은 표현형 적으로 원형벼와 동일했다. CPR 종자의 크기와 무게, 또한 생산량은 원형 벼와 비교하여 거의 차이가 없었다. 카페인의합성은 벼에서 생산되는 전구체, xanthosine의 양에 민감하게 좌우되었다.CPR 의 xanthosine과 카페인의 합성양은 8 배엽 단계에서 상관적으로 크게 증가하였으며 Xoo 와 M. oryzae에 대한 완전한 저항력을 갖추었다. 또한 CPR 은 벼 멸구 뿐만 아니라 가뭄 및 염스트레스에 대한 저항성이 강화되었다. 나는 카페인이 궁극적으로 살리실산의 합성을 활성화시키는 일련의 신호 전달 작용을 시작하는 분자 경로를 확인하였다. 이 결과는 방어 프라이밍 (defence priming)이라 불리는 병에 대한 대처법의 새로운 형태를 통하여 생물학적 및 비 생물학적 스트레스에 높은 수준의 내성을부여/제공하는 효과적인 수단임을 시사한다. 이는 벼뿐만 아니라 다른 작물 품종에 대한 장래의 분자육종에 잠재적으로 가치 있는 방어프라이밍을 위한 새로운 전략이 될 수 있다.

다국어 초록 (Multilingual Abstract)

Crop plants are constantly exposed to environmental factors, including biotic / abiotic stresses, which severely reduce yields. To cope with such stresses, chemical pesticides have commonly been used, but their excessive application has often caused environmental problems. It is thus urgent to find an alternative ways that are safer and more effective than conventional procedures. Here I report that caffeine elevates the basic immunity in rice through transgenic approach. Caffeine (1, 3, 7-trimethylxanthine) is a typical secondary metabolite of purine alkaloids and is synthesized in a few plant species such as coffee, cacao, and tea. Caffeine is thought to protect host plants by its toxicity against pathogens and herbivores. Caffeine is synthesized using a precursor, as the xanthosine in purine catabolism pathway. A caffeine-producing rice (CPR) was then generated by introduction of three N-methyltransferase genes identified from Coffea arabica. CPR plants were phenotypically equal to a wild type. The size and weight of the CPRs seed, also yields almost no difference as compared to the wild type. Caffeine synthesis sensitively depends on amount of precursor, xanthosine produced in rice. Xanthosine and caffeine amounts in CPR are correlatively increased at eight-leaf stages and the CPR has full resistance to Xoo and M. oryzae, at eight stages. In addition, CPR plants showed enhanced resistance not only to white backed plant hoppers but also to drought and salt stresses. The molecular pathway was identified through which caffeine initiates a series of signal cascade that ultimately activates synthesis of salicylic acid. These results suggest that caffeine is an effective means of conferring / provide a high level of resistance to biotic and abiotic stresses through a new form of disease coping method called defense priming. This can be a novel strategy for defense priming, being potentially valuable for future molecular breeding of not only rice but also other crop varieties.

목차 (Table of Contents)

• Introduction １
• Materials and Methods ３
• Transgenic rice plants and growth condition ３
• Bacterial and fungal strains and insect ３
• Anti-bacterial and anti-fungal assays of caffeine ４
• Bactericidal effect of caffeine ４
• Caffeine feeding into rice and Xoo inoculation ５
• RNA isolation, RT-PCR and quantitative RT-PCR analysis ５
• Determination of caffeine and xanthosine levels in rice leaves ６
• Determination of grain yield ７
• Bacterial population in planta ７
• M. oryzae inoculation and disease evaluation ７
• Insect repellence test ７
• Assay for phosphodiesterase activity ８
• Determination of cAMP contents in rice leaves ８
• Fluorescence imaging of intracellular Ca2+ ９
• Recombinant protein expression and purification ９
• Autokinase and Phospho-transfer assay １０
• Determination of SA and SAG levels in rice leaves １１
• Drought treatment １２
• Determination of H2O2 levels in rice leaves １２
• Assay for activity of antioxidant enzymes １２
• Salt treatment １３
• Statistical analysis １４
• Results １５
• Direct effect of caffeine on growth inhibition of pathogenic bacterial and fungal １５
• Resistance to bacterial leaf blight by exogenous treatment of caffeine １５
• Characterization and validation of caffeine producing rice １６
• Resistance to broad range of biotic stresses in CPR １７
• Signal transduction induced by caffeine in CPR １７
• Drought stress tolerance in CPR １９
• Discussion ２０