Previous studies showed that diverse genes function in spotted leaf phenotype. SPL11 protein includes a U-box domain and an armadillo (ARM) repeat domain, which were respectively functioned in yeast and mammalian to be taken part in ubiquitination and protein-protein interaction [19]. SPL28 occurs to be involved in the control of vesicular trafficking, and impaired SPL28 causes the initiation of hypersensitive response (HR)-like lesion, causing the leaf senescence [20]. Furthermore, the formation of spotted leaf is related to highly accumulation of ROS. When ROS production surpasses the scavenging capacity, ROS act as cytotoxins to generate necrotic lesions, leading to accelerated cell death [78, 79]. Plants overcome oxidative stress with the generation of scavenging enzymes such as catalases decomposing H2O2. In Arabidopsis, CAT1 is controlled by ABA, and MKK1-MPK6 regulate H2O2 metabolism via CAT1 [80]. Moreover, MEKK1, the Arabidopsis MAPK kinase kinase, knockout plant accumulates high level of ROS and develops local lesion similar with programmed cell death (PCD) (Teige et al., 2004). A role for MAPK cascades has been researched during
40
overexpression plants displayed spontaneous hypersensitive response (HR)-like spot on leaves, accumulation of defense-related genes and upregulation of phenolic compounds and phytoalexins. As a result, these transgenic plants gained enhanced resistance to a Magnaporthe grisea [43].
OsEDR1-suppressing / knockout plants have enhanced resistance to
bacterial blight disease caused by Xanthomonas oryzae pv. oryza (Xoo).
This resistance was connected to accumulation of salicylic acid (SA) and jasmonic acid (JA), expression of SA- and JA- related genes and inhibited accumulation of ACC, the direct precursor of ethylene, and expression of ethylene related genes [47].
In this study showed that two kinds of ROS such as H2O2 and singlet oxygen were highly accumulated in spl3 mutant under continuous light condition displaying reddish brown spot on the leaves surface (Fig.2).
Furthermore, under ABA-containing media spl3 mutant showed less defect of root development compared to wild type(Fig. 5), suggesting that SPL3 regulates ABA-mediated signaling via phosphorylation of MAPK cascades or unknown protein.
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초록
이 연구는 잎에 적갈색의 반점이 생기는 spotted leaf 3 돌연변이체를 이용하여 spl3 유전자를 지도기초분리 (Map-based cloning) 하고, 다양한 비생물학적 스트레스 처리 결과 나타나는 spl3 유전자의 분자 생물학적 기능을 나타낸 논문이다. 이러한 표현형은 식물체의 분얼기 이후 오래된 잎에서부터 적갈색 반점이 시작된다. spl3 돌연변이체는 지속적 광조건 (continuous light)을 처리했을 때 잎의 반점이 일반 조건에서 자란 식물보다 빠르게 생성되는 것을 확인하였다. 이러한 반점이 생성될 때 활성산소(ROS)의 생성량을 측정해보고자 DAB staining 과 SOSG 를 실시하였는데 그 결과 정상벼(wildtype)보다 spl3
돌연변이체에서 활성산소가 더 많이 축적된 것을 알 수 있었다. spl3 의
단순열성표현형을 조절하는 유전자를 찾기 위해 지도기초분리 (map-based cloning)를 실시하였고, 최종적으로 좁혀진 161.2kb 의 후보 지역에서 12 개의 후보 유전자가 존재하였고, 확인 결과 MAPK kinase kinase(MAPKKK)를 코딩하는 Os03g06410 유전자의 첫 번째 exon 의 1 개의 염기가 결실된 deletion mutation 이 되었음을 발견하였다. spl3 유전자가 다양한 비생물학적 스트레스의 신호 전달에 관여하는 MAPKKK 의 역할을 하기 때문에 정상벼에서 비생물학적 스트레스를 처리 한 이후 시간별로 SPL3 유전자의 발현을 확인하였다. 또한 SPL3 유전자의 부위별 발현량을 확인하였는데 잎에서 발현량이 가장 많음을 알
50
정상형보다 ABA 호르몬에 저항성을 가지는 표현형을 보였다. 잎에서도 chlorophyll level 이 spl3 돌연변이와 T-DNA Knockout 식물체에서 정상형보다 높은 수준으로 유지되는 것을 확인하였다. ABA insensitive 표현형을 가지는 유전자들의 발현량을 측정하였는데 ABI1 과 ABL1 의 유전자가 ABA 처리전에는 비슷한 발형량을 보였지만 ABA 처리 후 정상형 식물체에서는 발현량이 증가하고 spl3 돌연변이체에서는 상대적으로 적은 발현량을 보였다. 이것은 SPL3 단백질이 MAPKKK 이기 때문에 하위에 존재하는 MAPKK 나 MAPK 를 통한 신호 전달을 통하거나, 본 연구에서 밝혀지지 않은 다른 신호 전달 체계를 거쳐 ABI1 혹은 ABL1 유전자의 발현량을 조절 한다는 결과로 판단된다. 따라서 이 논문은 spotted leaf 3 와 같이 잎에 적갈색의 반점이 보이는 돌연변이체 연구에 중요하게 쓰일 것으로 기대되며 나아가 MAPKKK 의 비생물학적 스트레스, 특히 ABA 와 관련된 새로운 생리학적 기능을 제시한다.