Molybdenum (Mo) is an essential trace element for living organisms; however, molybdate transporter has not been identified in eukaryotes. The present thesis describes the first identification of a gene encoding eukaryotic molybdate transporter, MOT1, from Arabidopsis thaliana and the characterization of MOT2, a MOT1-like gene. MOT1 transcript is accumulated in both roots and shoots. The growth of mot1-1 and mot1-2 mutants was suppressed under Mo limited conditions, suggesting that MOT1 is essential for efficient molybdate uptake from soils. Molybdate uptake assay using yeast expressing MOT1 revealed that MOT1 is a high-affinity molybdate transporter. The analysis of a transferred DNA insertion line for MOT2 suggested that MOT2 is involved in molybdate translocation from roots to shoots. This thesis provides the basis for understanding the molecular mechanisms of Mo transport in plants.

Introduction

Molybdenum (Mo) is an essential element for all living organisms. Mo is a transition element, and is used by several enzymes that participate in reduction and oxidation reactions. In molybdenum-requiring enzymes (molybdoenzymes), except for bacterial nitrogenase, Mo is bound to pterin as a form of Mo-cofactor (Moco). In plants, four enzymes, nitrate reductase, aldehyde oxidase, sulfite oxidase, and xanthine oxidase are known as molybdoenzymes. Nitrate reductase catalyzes reduction of nitrate to nitrite, the first step of nitrate assimilation to ammonia and amino acids. Aldehyde oxidase is involved in an oxidation reaction that leads to the synthesis of abscisic acid. Plants that are incapable of using Moco are shown to be defective in nitrate reduction and abscisic acid biosynthesis.

Plants take up Mo from soil as molybdate (MoO42-). Molybdate is a weak Lewis acid, and the availability of Mo depends on soil pH. Mo deficiency is a widespread agricultural problem, especially in acid soils.

Regarding to the mechanism of Mo uptake from environment, molybdate transporters (ModABC), which belong to the ATP-binding cassette protein superfamily, have been described in eubacteria and archaea. The ATP-binding cassette protein superfamily exists in eukaryotes; however, molybdate-specific transporters in eukaryotes have never been reported. In this thesis, I identified the first eukaryotic molybdate transporter as the causal gene of the difference of Mo concentrations in Arabidopsis thaliana two accessions, Col-0 and Ler. Identified transporter was not an ATP-binding cassette type protein.

Chapter 1An Arabidopsis thaliana high-affinity molybdate transporter required for efficient uptake of molybdate from soil

Dr. Takano determined the concentrations of 10 elements in shoots of A. thaliana plants grown hydroponically in a standard medium and noticed that the concentrations of Mo, but not the other nine elements examined, differed by approximately 3-fold between the accessions Col-0 and Ler. Based on the results of quantitative trait loci mapping of Mo concentration carried out by Dr. Takano, I mapped the causal gene of the difference in Mo concentration in shoots between the two accessions in detail. I identified the causal gene (At2g25680) that encodes a sulfate transporter-like protein, and named this gene MOT1 (molybdate transporter 1).

Analysis of the nucleotide sequences of this intronless MOT1 gene in Col-0 and Ler identified two differences; single nucleotide substitution resulting in amino acid sequence change, and deletion of a 53-bp sequence just upstream of the initiation codon of MOT1 in Ler. The MOT1 transcripts were detected in both roots and shoots, and molybdate induced their accumulation. In the transgenic plants expressing β-glucuronidase (GUS) under the control of the MOT1 promoter, strong GUS signal was observed in the endodermis and stele. In tobacco cultured cells transiently expressing green fluorescent protein-MOT1 (GFP-MOT1) fusion protein, GFP-MOT1 fusion protein was localized, in part, to plasma membranes and to vesicles.

Mo concentrations in both roots and shoots of the mutants carrying transferred DNAs in the coding region (referred to as mot1-1) and promoter region (referred to as mot1-2) of MOT1, were less than 30% of that in wild-type plants. When compared with fresh weight of the mot1-1 and mot1-2 plants grown on the standard medium supplemented with 170 nM molybdate, fresh weight on the standard medium without molybdate supply was reduced to 35% and 80%, respectively. On the other hand, fresh weights of wild-type plants grown on standard medium supplied with or without molybdate were not significantly different. These results indicate that MOT1 is required for efficient uptake and/or translocation of molybdate and for normal growth under conditions of limited molybdate supply.

Kinetics studies in yeast revealed that the Km value of MOT1 for molybdate is approximately 20 nM. Furthermore, molybdate uptake by MOT1 in yeast was not affected by coexistent sulfate, and MOT1 did not complement a sulfate transporter-deficient yeast mutant strain. These data confirmed that MOT1 is specific for molybdate and suggest that the high affinity of MOT1 allows plants to obtain scarce Mo from soil.

Chapter 2Arabidopsis thaliana molybdate transporter 2 (MOT2) is required for efficient translocation of molybdate

Homology search in the sequence databases suggests presence of genes similar to MOT1 in plants and fungi. At1g80310 is the most similar (Identity, 51%; Similarity, 82%) to MOT1 in Arabidopsis genome at amino acid sequence level and named MOT2. In this chapter, I examined whether MOT2 is involved in the transport of molybdate.

MOT2 is expressed both in roots and shoots. Molybdate did not induce MOT2 transcript accumulation. In the transgenic plants expressing GUS under the control of the MOT2 promoter, strong GUS signal was observed in the veins of leaves. In the cells of roots of the transgenic plants expressing GFP-MOT2 under the control of the cauliflower mosaic virus 35S RNA promoter, GFP-MOT2 fusion protein was localized to plasma membranes.

When plants grown on the standard medium without molybdate supply, the growth of the mutant plants carrying transferred DNA in the coding region of MOT2 (mot2-1) were reduced to less than 40% of that of wild-type plants. Mo concentrations in shoots of mot2-1 mutant plants grown on standard medium supplemented with 170 nM molybdate were less than 45% of that of wild-type plants. However, Mo concentrations in roots of mot2-1 mutant plants were approximately 2-fold higher than that of wild-type plants. Furthermore, in plants grown on standard medium without molybdate supply, Mo concentrations in shoots of mot2-1 mutant plants were not different from that of wild-type plants, although the shoot growth of mutants was suppressed. Taken together, these results suggest that MOT2 is involved in molybdate translocation from roots to shoots in A. thaliana.

Conclusion

MOT1 is a molybdate importer expressing in both roots and shoots. MOT1 is a high-affinity and molybdate specific transporter for taking up scarce molybdate from soil efficiently. In the shoot, MOT1 is expressed in various tissues.

MOT2 is expressed in both roots and shoots, especially in the veins of leaves. The phenotype of mot2-1 mutant suggests that MOT2 is essential for molybdate translocation from roots to shoots, efficiently.

Taken together, I established that A. thaliana has at least two types of molybdate transporters; one for uptake (MOT1-type) and the other for translocation (MOT2-type) of molybdate.

Figure 1 Transport properties of MOT1. (A) Time course of molybdate uptake in S. cerevisiae cells expressing MOT1 (open circles) or cells containing the empty vector (filled circles). The cells cultured with medium without molybdate were incubated with medium containing 170 nM molybdate for various times. Mo concentrations in cells were measured. (B) Lineweaver-Burk plot of molybdate uptake in S. cerevisiae cells expressing MOT1 cultured with medium supplemented with 7, 8, 10, 12, 16, 24, 97, or 194 nM molybdate for 15 min.

Figure 2 Mo concentrations in roots and shoots. Plants were grown for 3 weeks with medium containing 170 nM molybdate. mot2-1 and mot1-1 are mutant plants that carry transferred DNA in the coding region of At1g80310 (mot2-1) or At2g25680 (mot1-1). Averages and standard deviations are shown; n = 5.

モリブデン(Mo)は生物の必須元素であり、植物はMoを含む酵素により硝酸の同化、植物ホルモンの生合成、硫黄の代謝などを行っている。植物は土壌からMoを二価の陰イオンであるモリブデン酸(MoO4(2-))の形態で吸収すると考えられている。モリブデン酸は弱酸であり、植物のMo利用効率は土壌のpHに依存する。酸性土壌におけるMo欠乏は農業上の問題となっている。本論文は、モリブデン酸トランスポーターを真核生物より初めて同定したものである。

本論文は、序章に続く2つの章と結語からなる。

序章では植物のモリブデン研究の状況について述べている。

第1章では、シロイヌナズナのCol-0とLet間でのMo濃度の差を利用して、硫酸イオン輸送体類似タンパク質をコードする遺伝子(At2g25680)をモリブデン濃度を決定する原因遺伝子として同定しており、この遺伝子をMOT1(molybdate transporter1)と名付けている。塩基配列の比較により、LerのMOT1にはアミノ酸置換を伴う1塩基の置換と翻訳開始コドン上流に53塩基の欠失があることを確認している。MOT1転写産物は地上部と根で検出され、その蓄積は培地のモリブデン酸により誘導されることを示している。MOT1プロモーター制御下でβ-glucuronidase(GUS)を発現する形質転換植物では、根の中心柱付近、ロゼット葉の葉柄、花の雄しべとがく、英の外縁部において強いGUSシグナルが観察され、。緑色蛍光タンパク質(GFP)とMOTIの融合タンパク質をカリフラワーモザイクウィルス35SRNAプロモーター制御下で一過的に発現させたタバコ培養細胞では、細胞の外縁部と内部のにドット状に蛍光が観察された。さらに、MOT1の翻訳領域(mot1-1)またはプロモーター領域(mot1-2)に外来遺伝子が挿入された変異株では、地上部と根のMo濃度が野生型株の30%以下に低下していることを示し、Mo欠乏環境下での生育をMo充分環境下での生育と新鮮重で比較したところ、野生型では有意な差はなかったが、motl1-1変異株とmotl1-2変異株ではそれぞれ35%と80%に低下していたことを示している。また、MOTlを発現させた酵母は細胞内Mo濃度が上昇し、そのモリブデン酸イオン輸送の垢値は約20nMであった。この輸送は硫酸イオンによる競合的な阻害を受けなかった。また、MOTlは硫酸イオン輸送体欠損酵母を相補しなかった。これらの結果から、MOTlがモリブデン酸イオン特異的な高親和型トランスポーターであることを示している。

第2章においては、MOTlに相同な遺伝子の解析を行っている。Atlg80310はシロイヌナズナの遺伝子の中でMOTIとのアミノ酸配列の類似性が最も高い遺伝子である(同一性51%、類似性82%)。この遺伝子もまたモリブデン酸イオン輸送体をコードしていると推測し、MOT2と名付けた。MOT2転写産物は地上部と根で検出され、その蓄積はモリブデン酸による誘導を受けなかった。MOT2プロモーター制御下でGUSを発現する形質転換植物では、根の全体、ロゼット葉の葉脈と出水器官、花の雄しべとがく、英の外縁部においてGUSシグナルが観察された。カリフラワーモザイクウィルス35SRNAプロモーター制御下でGFPとMOT2の融合タンパク質を発現する形質転換植物の根では、細胞の外縁部の一部においてGFPの蛍光と細胞膜マーカーFM4-64の蛍光との共局在が観察された。MOT2の翻訳領域に外来遺伝子が挿入された変異株(MOT2-1)のMo欠乏環境下での新鮮重は野生型株の40%以下に低下していた。Mo充分環境下において、MOT2-1変異株の地上部のMo濃度は野生型株の45%以下に低下していた。一方で、MOT2-1変異株の根のMo濃度は野生型株の約2倍に上昇していた。このとき、根におけるMOT1転写産物の蓄積量はMOT2-1変異株と野生型株で有意な差がなかった。これらの結果を基に、MOT2が根から地上部へのモリブデン酸イオンの転流に関与していることを結論付けている。

以上、本論文は、真核生物のMo輸送体を初めて同定し、植物においてモリブデン酸イオン輸送が硫酸イオン輸送とは異なる輸送体によって行われる一例を示したものであり、植物の輸送研究分野に、極めて高い貢献をしている。

よって、審査委員一同は、本論文を博士論文として高く価値あるものと認めた。