Date: 2018-08-16 (Thu)

アラビドプシスのCRF1(Constitutive Response to fe-deficiency 1)の同定とその性質

Poster at ISINIP in Taiwan 2018

植物が土壌から鉄を吸収して各組織に移行させる分子成分やシグナルのネットワークを包括的に明らかにする必要がある。これまでに知られている鉄獲得に関わる因子は大部分がポジテイブレギュレーターで、ネガテイブレギュレーターの報告は僅少である。そこで鉄ホメオスタシス(生体内調和)に関わるネガテイブレギュレーターを同定するためにIRT1(iron regulated transporter-1)プロモーター駆動下のluciferaze (PIRT1:LUC)というコンストラクトを作成して、遺伝子のスクリーニングを行った。このレポーターシステムは鉄欠乏下2−3日で発現し、鉄添加で発現は抑制された。つぎに遺伝子変異源であるEMS(Ethyl Methane Sulfonate)でこのreporter系統の種子を処理したのち、その変異株のスクリーニングを行い、あたらしい鉄ホメオスタシスに関する遺伝子CRF1(Constitutive Response to Fe-deficiency 1 )を同定した。変異株crf1は鉄欠乏、亜鉛過剰、カドミウム過剰、銅過剰、コバルト過剰、ニッケル過剰などに耐性で野生型(WT)よりも多くの鉄を集積した。変異株crf1では鉄欠乏誘導性遺伝子IRT1, FRO2(ferric reduction oxidase 2), FIT(Fer like iron-deficiency induced transcription factor), bHLH100/101/38/39らは恒常的に発現していた。遺伝子発現分析の結果 crf1-1株が興味ある高発現株であることが分かった。CRF1タンパクは重金属による毒性環境下でも生息できることに寄与できる鉄ホメオスタシスシグナル伝達系の上流に位置する調節因子である可能性が高い。

Identification and characterization of CRF1, Constitutive Response to Fe-deficiency 1 in Arabidopsis thaliana
Reena Sharma1,2,3 and Kuo-Chen Yeh*1,2,4
1Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia
Sinica and National Chung Hsing University, Taipei-11529, Taiwan
2Agricultural Biotechnology Research Center, Academia Sinica, Taipei-11529, Taiwan
3Graduate Institute of Biotechnology, National Chung Hsing University, Taichung-40227 Taiwan
4Biotechnology Center, National Chung Hsing University, Taichung-40227, Taiwan

Iron (Fe) is essential element for living organisms. Plants are the principal source of dietary Fe. Plant roots hunt soil for Fe and Fe concentration can be dramatically lower than required for growth. Its deficiency leads to developmental defects and excess can cause toxicity. Plants tightly control the iron dependent transcription to ensure the optimal Fe absorption. The molecular components and signalling networks involved in Fe acquisition from soil and transportation to destitute parts of plants are required to be studied comprehensively. Majority of the known factors involved in Fe acquisition are positive regulators but only one known negative regulator is reported so far. In order to identify negative regulators involved in Fe homeostasis, IRT1 (IRON REGULATED TRANSPORTER 1) promoter driven luciferase (PIRT1:LUC) construct was used for genetic screening. This reporter system is activated within 2-3 days under Fe deficient conditions and completely repressed under Fe sufficient conditions. EMS (Ethyl Methane Sulfonate) mutagenesis approach was used toscreen novel candidates. Constitutive Response to Fe-deficiency1 (crf1) mutant was identified with constitutive IRT1 transcript and protein expression despite of Fe status. The Fe specific mutant crf1 is tolerant to Fe deficiency, excess zinc, cadmium, copper, cobalt and nickel and can accumulates more Fe than wild type (WT). Quantitative analysis for Fe accumulation showed that in excess Cd and Zn, the Fe content is higher in crf1 than WT. Several Fe deficiency responsive genes including IRT1, FRO2 (FERRIC REDUCTION OXIDASE 2),
FIT (FER LIKE IRON-DEFICIENCY INDUCED TRANSCRIPTION FACTOR) and bHLH100/101/38/39 are constitutively expressed in crf1. Transcriptomic analysis has revealed interesting candidates involved in Fe deficiency response and heavy metal response and found to be highly expressed in mutant. CRF1 can help to plant survival under heavy metal toxicity and CRF1 could be a sensor and an upstream regulator in Fe
homeostasis signaling pathway.
Keywords: Forward genetics, Fe deficiency response, Fe accumulation, Heavy metal tolerance, Arabidopsis thaliana