Functional analysis of transcription factors that are phosphorylated by MAP kinases in Arabidopsis

다국어 초록 (Multilingual Abstract)

The mitogen-activated protein kinase (MAPK or MPK) pathways are ones of the most important and conserved signaling events in plants. MPKs directly modulate gene expression by phosphorylating their substrates such as transcription factors. Some Arabidopsis MPK-interacting transcription factors were isolated by high-throughput yeast two-hybrid screening. They are belonging to different families such as GRAS, AP2, bZIP, zinc finger, HSF and MYB proteins. Among them ERF5, ERF6, ERF8, MYB41, MYB44, MYB77, HSFA3 and ZAT10 transcription factors were phosphorylated by MPK3 and MPK6 in phosphorylation assays.
ZAT10 and MYB44 were selected for further functional analyses. Phosphorylation assays showed that ZAT10 and MYB44 proteins were not only phosphorylated by recombinant MPK3/6 but also by plant extracted MPK3/6. By MALDI-TOF mass spectrometry S8 and S210 of ZAT10, and S53 and S145 of MYB44 were identified as MPKs phosphorylation sites, respectively. To confirm the specificity of these phosphorylation sites, serine residues were mutated to alanin by site-directed mutagenesis. MPK3/6 could phosphorylate original proteins (ZAT10WT/MYB44WT) but not phosphorylate mutated proteins (ZAT10AA/ MYB44AA).
ZAT10 was known as a key member of the C2H2-type zinc finger transcription factor family in Arabidopsis. Gene expression analyses indicated that ZAT10 is induced by environmental stresses. ZAT10 overexpressing transgenic plants exhibited enhanced tolerance to osmotic stress, but zat10 knockout mutant plants showed an osmotic stress sensitive phenotype. These results demonstrated that ZAT10 is a positive regulator in conferring osmotic stress tolerance in Arabidopsis. The zat10 mutant phenotype was complemented by the expression of ZAT10WT protein but not by the expression of ZAT10AA mutant protein, indicating that the phosphorylation of ZAT10 by MPKs is essential for the physiological function of ZAT10 in plant.
A member of Arabidopsis R2R3 MYB transcription factor family, MYB44, was known to be involved in various stresses such as dehydration, low temperature, salinity and ABA, ET or methyl jasmonate. Overexpressing MYB44 transgenic plants were enhanced the tolerance to salt and drought stresses via ABA-mediated signaling pathway. In this study, I show that the transcription of MYB44 is high accumulated in the dry seed as well as in the seedlings at low GA condition, and that transcription level is reduced in the germinating seeds. Decreased GA synthesis not only inhibits the germination of transgenic overexpressing MYB44 seeds but also enhances the germination of myb44 mutant seeds comparing to wilt-type. When two serine phosphorylation sites were mutated to alanin residues by site-directed mutagenesis, the phosphorylation of MYB44 by MPKs was abolished and the expression of MYB44AA mutant protein could not rescued the insensitive phenotype of myb44 mutant seeds to low endogenous GA and high exogenous ABA levels as MYB44WT protein did. Finally, MYB44 protein could be bound to ABI5-1 and ABI5-2 oligos which are containing the consensus MYB binding site (TAACTG) in ABI5 promoter. These results demonstrated that MYB44 transcription factor participate in the regulation of ABA and GA signaling to modulate the seed germination at transcriptional and post-translational levels in Arabidopsis.

국문 초록 (Abstract)

식물체가 외부 자극을 인식하여 다양한 신호전달 기작을 통해 세포 내의 여러 유전자의 발현을 조절함으로써 자극에 대한 세포 반응을 하게 된다. 이러한 신호전달 기작 중의 하나로서 Mitogen-activated protein kinase (MAPK or MPK) 매개 신호전달 경로가 잘 알려져 있다. MPK들은 전사조절인자 같은 그들의 기질을 직접적으로 인산화 (phosphorylation) 시킴으로써 유전자의 발현을 조절한다. 본 연구에서는 high-throughput yeast two-hybrid screening을 통해서 MPK와 상호작용하는 전사조절인자를 동정하였다. 그 결과 GRAS, AP2, bZIP, zinc finger, HSF와 MYB과 같은 다양한 전사조절인자 그룹들이 동정되었다. 인산화 분석 (Phosphorylation assay)을 이용하여 그들 중에서 MPK3/6에 의해 ERF5, ERF6, ERF8, MYB41, MYB44, MYB77과 ZAT10이 인산화된다는 것을 확인했다.
그들 중 ZAT10과 MYB44을 선택하여 이후의 실험들을 진행하였다. ZAT10과 MYB44는 recombinant MPK3/6 뿐만 아니라 식물세포에서 추출된 MPK3/6에 의해서도 인산화된다는 것을 알 수 있었다. MALDI-TOP mass spectrometry를 통해 ZAT10의 S8과 S210, MYB44의 S53과 S145이 MPK에 의해 인산화되는 잔기라는 것을 확인했다. 인산화 잔기를 검증하기 위해 site-directed mutagenesis을 통해 serine 잔기를 alanine으로 치환하였다. 그 결과 ZAT10과 MYB44의 original protein은 MPK3/6에 의해 인산화 되는 반면 mutation시킨 protein (ZAT10AA/MYB44AA)은 인산화 되지 않음을 알 수 있었다.
ZAT10은 애기장대에서 C2H2-type zinc finger transcription factor family로 잘 알려져 있으며 다양한 스트레스에 의해 ZAT10 유전자의 발현이 증가한다고 알려져 있다. ZAT10의 생리학적 기능을 규명하고자 다양한 스트레스를 처리한 후 ZAT10 형질 전환체와 zat10 돌연변이 식물체의 표현형을 관찰하였다. 그 결과 ZAT10 과발현 형질전환 식물체와 zat10 돌연변이 식물체는 각각 osmotic stress에 대해 저항성과 민감성 표현형을 나타냄을 알 수 있었다. 실험 결과로서 ZAT10은 애기장대에서 osmotic stress tolerance에 positive regulator라는 것을 확인 할 수 있었다. Complementation 실험을 통해 ZAT10AA mutant protein이 zat10 돌연변이 표현형을 회복 시키지 못함을 확인함으로써 MPK에 의한 ZAT10 단백질의 인산화가 그 기능에 필수적임을 보여주었다.
MYB44는 애기장대에서 R2R3 MYB transcription factor family에 속한다. MYB44는 건조, 저온, 고염, ABA, ET 그리고 MeJ 같은 다양한 스트레스에 관여한다고 보고되어 있다. 본 연구에서 seed germination 동안에 MYB44의 transcript가 줄어드는 것을 확인하였다. 흥미롭게도 ABA가 함유된 배지에서 종자발아 분석을 통해 WT과 myb44 돌연변이에 비해 MYB44 과발현 형질전환 식물체가 ABA에 과민한 표현형을 가짐을 확인 하였다. 저해제를 처리하여 GA 합성을 감소시키면 MYB44 과발현 형질전환 식물체의 종자는 발아율이 줄어들고, 반면에 myb44 돌연변이 식물체 종자 발아율이 증가함을 확인하였다. Complementation 실험을 통해 인산화 되지 않는 MYB44 돌연변이 단백질 (MYB44AA)가 myb44 돌연변이 식물체 표현형을 회복시키지 못함을 보여 줌으로써 MYB44의 MPK에 의한 인산화가 그 기능에 필수적임을 보여주었다. 그리고 MYB44 protein은 consensus MYB44 binding site (TAACTG)이 포함된 ABI5 promoter인 ABI5-1과 ABI5-2에 결합한다는 것을 EMSA assay를 통해 확인하였다. 본 연구를 통하여 MYB44는 종자 발아의 억제자로 기능을 하며 GA에 의해서는 그 발현이 감소하며, ABA에 의해서는 MYB44 단백질이 MPK에 의하여 인산화되어 활성화되어 종자 발아를 억제할 것으로 예상된다.

목차 (Table of Contents)

• I. Introduction 1
• II. Materials and methods 9
• 1. Plant materials 9
• 2. DNA gel blot analysis 9
• 3. Genomic DNA-PCR and RT-PCR analyses 9
• 4. Plant growth conditions, seed germination assay, and stress treatments 10
• 5. Yeast two-hybrid (Y2H) screening 11
• 6. Protein extraction from Arabidopsis 11
• 7. Western blot analysis 12
• 8. Preparation of recombinant proteins 12
• 9. Pull-down binding assay 13
• 10. Luciferase complementation imaging (LCI) assay 13
• 11. Site-directed mutagenesis 14
• 12. Kinase assay 14
• 13. In-gel kinase assay 15
• 14. Mass spectrometry 15
• 15. Generation of transgenic plants 16
• 16. Electrophoretic mobility shift assay 16
• III. Results 17
• 1. Identification of transcription factors as the substrates of MPKs 17
• 1.1. Isolation of MPK-interacting transcription factors 17
• 1.2. Phosphorylation of isolated transcription factors by MPK3 and MPK6 17
• 2. Biochemical properties of ZAT10 and its functional analysis in Arabidopsis 21
• 2.1. Interaction of ZAT10 and MPK3 and MPK6 in vitro and in planta 21
• 2.2. Phosphorylation of ZAT10 by MPK3 and MPK6 23
• 2.3. Identification of two phosphorylation sites of ZAT10 by mass spectrometry 25
• 2.4. Verification of the phosphorylation sites of ZAT10 by site-directed mutagenesis 25
• 2.5. Transcriptional analysis of ZAT10 under various stress conditions 30
• 2.6. The biological function of ZAT10 in osmotic stress tolerance 30
• 2.7. Changes of expression of stress responsive genes in the zat10 mutant 37
• 2.8. Functional analysis of EAR domain in ZAT10 37
• 2.9. Regulation of phosphorylation of ZAT10 by MPKs in osmotic stress response 38
• 3. Biochemical properties of MYB44 and its functional analysis in Arabidopsis 43
• 3.1. Phosphorylation of MYB44 by MPK3 and MPK6 43
• 3.2. Identification of two phosphorylation sites of MYB44 by mass spectrometry 45
• 3.3. Verification of the phosphorylation sites of MYB44 by site-directed mutagenesis 45
• 3.4. Reduction of MYB44 transcripts during the seed germination 49
• 3.5. The biological function of MYB44 in GA-induced seed germination 49
• 3.6. Effect of the MPKs phosphorylation sites of MYB44 in seed germination 56
• 3.7. Binding properties of MYB44 to the consensus MYB binding site (TAACTG) in the ABI5 promoter 59
• IV. Discussion 61
• 1. Arabidopsis transcription factors were identified as substrates of MPK3 and MPK6 in vitro 61
• 2. ZAT10 is a positive regulator of osmotic stress tolerance in Arabidopsis 62
• 3. The phosphorylation of ZAT10 by MPKs plays a key role in its physiological function in Arabidopsis 64
• 4. MYB44 transcription factor modulates a GA signaling pathway that helps to regulate seeds germination in Arabidopsis 66
• 5. MPKs phosphorylation sites of MYB44 is important for Arabidopsis seed germination under low-GA condition 68
• V. References 73
• VI. Acknowledgements 84