초록 ▼

Ⅳ. 연구개발결과
BOI 단백질군이 DELLA 단백질과 결합하여 지베를린에 의해 촉진되는 phase transition 과정을 억제한다는 것을 다양한 돌연변이체를 사용하여 증명하였다. 또한 분자수준에서는 BOI가 DELLA 의존적으로 DELLA가 결합하는 프로모터에 타겟팅되어 유전자 발현을 억제한다는 것을 보였다. 마지막으로 BOI가 개화를 CO 의존적 및 DELLA 의존적 방법에 의해 조절한다는 것을 분자유전학적 분석을 통해 밝혔다.
BR 신호전달 과정의 중요 전사인자인 BES1과 BZR1이 ABA2 유전자 발현을 촉진

Ⅳ. 연구개발결과
BOI 단백질군이 DELLA 단백질과 결합하여 지베를린에 의해 촉진되는 phase transition 과정을 억제한다는 것을 다양한 돌연변이체를 사용하여 증명하였다. 또한 분자수준에서는 BOI가 DELLA 의존적으로 DELLA가 결합하는 프로모터에 타겟팅되어 유전자 발현을 억제한다는 것을 보였다. 마지막으로 BOI가 개화를 CO 의존적 및 DELLA 의존적 방법에 의해 조절한다는 것을 분자유전학적 분석을 통해 밝혔다.
BR 신호전달 과정의 중요 전사인자인 BES1과 BZR1이 ABA2 유전자 발현을 촉진해서 ABA 함량을 낮춘다는 것을 증명하였고, ACO2/ACO4 발현을 억제한다는 것을 보였고, 옥신 신호전달의 중요 전사인자인 ARF7이 BAS1 유전자 발현을 억제한다는 것을 증명하였다.
BAK1이 공변세포에 존재하고, bak1 돌연변이체가 ABA-induced stomatal closure 현상에 대해서 ABA-insensitive한 반면, H2O2가 직접적으로 처리되었을 때에는 정상적인 공변세포의 개폐 현상을 보이는 것은 BAK1이 최소한 H2O2가 관여하는 단계의 상위에서 ABA signaling을 통한 공변세포의 개폐현상을 조절하는 인자임을 의미한다. 본 연구진은 당해연도에 기존에 알려져 있었던 stomatal signaling의 중요 component인 RPK1과 BAK1과의 상호 작용을 보다 정교한 비교구를 포함한 실험으로 입증하였고, RPK1이 BAK1에 의해 인산화됨을 밝혔다. 또한 BAK1 추가적인 기능을 탐색하기 위하여 본 연구진이 bak1 돌연변이체를 대상으로 activation tagging approach를 통한 suppressor screening을 수년전부터 수행해오고 있었고, 당해연도에 putative suppressor를 발굴하였고, miR172의 과발현에 의해서 bak1의 표현형이 전생애에 걸쳐서 억제됨을 알 수 있었다.
ethylene 신호체계와 GLIP1의 기능적 상관관계를 규명하고자 Y2H screening을 통해 관련된 조절인자와 전사인자들을 확보하고 이들의 기능과 상호작용 기작을 연구하였다. 또한 GLIP1 유래 유도면역 조절 신호 대사물질을 발굴, 확보하고자 GLIP1과 ESM1의 상호작용을 조사하고, 이들 과발현체의 체관수액을 분석하여 특이적 대사물질을 동정하였다.
종자발아 조건에서 RNA-seq을 수행한 후, phy-PIL5에 의해서 억제되고 red light에 의해 증가하는 전사인자를 발굴한 후에, SOM이 프로모터에 결합하는 전사인자를 발굴하였고, 돌연변이체 및 형질전환체를 사용하여 발아관련 형질을 분석하였다. 이를 통해 C3라고 이름붙인 전사인자가 후성유전학적 요소인 JMJ20과 결합하여 발아를 조절한다는 것을 증명하였다.

Abstract ▼

DELLA proteins, consisting of GA INSENSITIVE, REPRESSOR OF GA1-3, RGA-LIKE1 (RGL1), RGL2, and RGL3, are central repressors of gibberellin (GA) responses, but their molecular functions are not fully understood. We isolated four DELLAi nteracting RING domain proteins, previously designated as BOTRYTIS

DELLA proteins, consisting of GA INSENSITIVE, REPRESSOR OF GA1-3, RGA-LIKE1 (RGL1), RGL2, and RGL3, are central repressors of gibberellin (GA) responses, but their molecular functions are not fully understood. We isolated four DELLAi nteracting RING domain proteins, previously designated as BOTRYTIS SUSCEPTIBLE1 INTERACTOR (BOI), BOI-RELATED GENE1 (BRG1), BRG2, and BRG3 (collectively referred to as BOIs). Single mutants of each BOI gene failed to significantly alter GA responses, but the boi quadruple mutant (boiQ) showed a higher seed germination frequency in the presence of paclobutrazol, precocious juvenile-to-adult phase transition, and early flowering, all of which are consistent with enhanced GA signaling. By contrast, BOI overexpression lines displayed phenotypes consistent with reduced GA signaling. Analysis of a gai-1 boiQ pentuple mutant further indicated that the GAI protein requires BOIs to inhibit a subset of GA responses. At the molecular level, BOIs did not significantly alter the stability of a DELLA protein. Instead, BOI and DELLA proteins are targeted to the promoters of a subset of GA-responsive genes and repress their expression. Taken together, our results indicate that the DELLA and BOI proteins inhibit GA responses by interacting with each other, binding to the same promoters of GAresponsive genes, and repressing these genes. In addition, we investigated how the BOIs repress flowering. Genetic analysis using the boiQ quadruple mutant indicated that BOIs repress flowering mainly through FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), as the ft soc1 boiQ hextuple mutant shows a late flowering phenotype similar to that of the ft soc1 double mutant. BOIs repress the expression of FT and SOC1 by CONSTANS (CO)-dependent and –independent mechanisms. This dual repression of FT and SOC1 makes BOIs strong repressors of flowering in both CO-dependent and CO-independent pathways in Arabidopsis thaliana.
Molecular regulation of endogenous level of plant hormones, especially ethylene, ABA and brassinosteroids (BRs) by BR signally was investigated in A. thaliana. In Arabidopsis roots, expression of ACC oxidase 1 (ACO1) which mediates conversion of ACC to ethylene in ethylene biosynthesis increased by exogenously applied BRs and in a BR dominant mutant, bes1-D. Electrophoretic mobility shift assay (EMSA) and chromatin-IP assay showed that BES1 directly binds to E-box sequences in prompter region of ACO1. GUS report system provided that expression of ACO1 is only detected by binding of BES1 to the E-boxes, indicating that BES1 directly regulates expression of ACO1. GC-MS analysis demonstrated that ethylene production is increased by binding of BES1 to the E-boxes in Arabidopsis roots, which promotes activation of growth and gravitropic response of Arabidopsis roots. Expression of ACO2 which was dominantly expressed in Arabidopsis shoots was down-regulated by exogenously applied BRs and in a BR dominant mutant, bzr1-1D. EMSA and chromatin-IP indicated that BZR1 binds E-boxes in the promotor region of ACO2. GC-MS analysis provided that binding of BZR1 to the promotor of ACO2 decreases endogenous level of ethylene in Arabidopsis shoots, which leads altered growth of Arabidopsis shoots.
ABA2 is a biosynthetic gene which catalyzes conversion of xanthoxal to ABA aldehyde in ABA biosynthesis. Real-time PCR demonstrated expression of ABA2 is down-regulated by BRs. EMSA and chromatin-IP assay suggested that both BES1 and BZR1 bind to E-boxes in the promotor region of ABA2, indicating that BR transcription factors directly regulate ABA2 expression. Radio-immunoassay and GC-MS analysis demonstrated endogenous amounts of ABA are reduced in a BR dominant mutant, bes1-D. Phenotype analysis of BR and ABA related mutants suggested that BR regulated ABA2 expression is important for germination, stress responses, embryogenesis and seed development of Arabidopsis.
Biochemical and molecular genetic analysis of DWARF1 verified that DWARF1 mediates conversion of 24-methylene BRs to 24-methyl BRs in Arabidopsis. Reduced expression of DWARF1 by downstream BRs and in bes1-D suggested that expression of DWARF1 is feedback regulated by BR signalling in the plant. EMSA revealed BES1 directly binds to E-box sequences in promotor region of DWARF1, indicating that the feedback regulation of DWARF1 is mediated by BES1. BAS1 is a catabolic gene in BR homeostasis in Arabidopsis. Expression of BAS1 is down-regulated by exogenously applied BRs. EMSA indicated that both BZR1 and an auxin transcription factor, ARF7 directly bind cis-elements in promotor region of BAS1. Yeast two hybrid assay and DNA pull down assay suggested BZR1 and ARF7 interact to express of BAS1, which implicates that BZR1 and ARF7 competitively regulate to maintain endogenous level of BRs in Arabidopsis.
BAK1 was originally identified as a co-receptor of BRI1, a BL-binding receptor to mediate signalings caused by brassinosterids that are one of the important plant hormones for plant growth and development. However, further research combined with biochemical and genetic approaches revealed that BAK1 functions various developmental processes, including plant defense and cell death control. In this study, we investigated additional functions of BAK1 in an ABA-induced stomatal movement Mutant lacking BAK1 showed higher transpirational water loss through stomata and reduced sensitivity to ABA in stomatal closure. BAK1 interacts with RPK1 specifically in the plasma memebrane of guard cells and phosphorylates the RPK1.
To identify more components involved with BAK1-mediated processes, we performed a suppressor screening of bak1 and found a gain-of-suppressor, bak1-SUP1, in which a miR172 was overexpresed. From a detailed phenotypic and genetic analyses, we concluded that the miR172 plays a positive role in growth. As miR172 was known to regulate flower organ development so far, our results added novel function of miR172 in vegetative development, too.
We also found that an another interesting aspect for BAK1 is phenotypes of the transgenic plants overexpressing BAK1 were changed by the balanced control of both growth and defense potentials. To further investigate underlying regulatory mechanisms, we found a genetic suppressor of BAK1 overexpressor using an activation–tagging method. T-DNA was inserted near in a gene encoding a cytoplasmic serine/threonie kinase, leading to the activation of this gene. The genes related with SA-dependent plant immunity were down-regulated in the suppressor.
Signaling is a central part of systemic resistance that requires a systemic signal. It is believed that a signal is generated at the primary infection site and translocated to the rest of plant tissues to activate defense mechanisms. A key step for understanding the signaling mechanism of systemic resistance is the identification of the systemic signals. Whereas recent evidence suggest critical roles for lipids in systemic resistance signaling, the nature of this signal and the underlying mechanism of systemic resistance has not been clearly determined.
Our recent studies demonstrate that GLIP1-elicited systemic resistance is dependent on ethylene signaling and provide evidence that GLIP1 mediates the production of a systemic signaling molecule(s). From close collaborative works with Metabolome Analysis Team (led by Dr. Myung Hee Nam) in Korea Basic Science Institute, we identified candidates for the mobile signaling molecule in the phloem of 35S:GLIP1 transgenic Arabidopsis plants. We made our effort to elucidate proteins and receptors interacting with GLIP1 in systemic resistance signaling. The signaling components in upstream and downstream signaling processes of GLIP1 were identified and studied for their interactions with GLIP1 and roles in systemic immunity. We also explored the molecular mechanisms underpinning the relationship between GLIP1 and ethylene signaling through an epistatic analysis of ethylene response mutants and GLIP1-overexpressing and mutant plants. We proposed a model explaining how GLIP1 regulates the fine-tuning of ethylene signaling and ethylene-SA crosstalk.
Our research on GLIP1, mobile signal, and their interacting proteins/receptors in SAR would provide insights into defense signaling pathways and their crosstalk, and lead to publications in leading-edge journals. The results would be conjugated to studies in other fields such as plant growth, development, and various stress responses. Furthermore, GLIP1, signaling molecules and other interacting factors identified in our studies would be ultimately applied to development of GM crops with enhanced pathogen resistance traits, providing good resources for biotechnological applications.
In a previous study, we showed that the histone arginine demethylases, JMJ20 and JMJ22, act redundantly as positive regulators of seed germination in the PHYB-PIF1-SOM pathway. Upon PHYB activation by germination-inducing red light, JMJ20/JMJ22 are de-repressed resulting in increased gibberellic acid (GA) levels through the removal of repressive histone arginine methylations at GA3ox1/GA3ox2, key genes for GA biosynthesis. Thus, JMJ20 and JMJ22 mediate light signal to GA biosynthesis and seed germination. However, it is not known how JMJ20 and JMJ22 are specifically recruited to the GA3ox1/GA3ox2 loci upon induction by light. In this study, first we found that a SOM-repressed factor(s) is responsible for the specific targeting of JMJ20/JMJ22 to GA3ox1/GA3ox2. Our RNA seq analysis allowed the identification of 5 genes (C1 to C5) encoding transcription factors that are repressed by SOM and derepressed by red light and imbibition. Subsequently, we found that only C3 among those is directly repressed by SOM. Consistently, c3 loss-of-function mutants showed delayed-germination phenotypes similar to that of the jmj20 jmj22 double mutant. Furthermore, the germination phenotype of c3 jmj20 jmj22 triple-mutant seeds was not distinguishable from the phenotypes of c3 and jmj20 jmj22 mutants seeds, indicating that C3 acts in the same genetic pathway to JMJ20/JMJ22. Finally, we demonstrate through an in vitro pull-down assay that C3 and JMJ20 proteins can interact each other. Therefore, C3 might be a transcription factor that allows the targeting of JMJ20/JMJ22 to GA3ox1/GA3ox2 during light-induced seed germination in Arabidopsis.