Plants are the producers with photosynthetic ability, and we humans are dependent on them. For example, plants can assimilate inorganic sulfate into cysteine and synthesize methionine, and methionine is an essential amino acid for humans. As land plants are dependent on soils, plants have evolved to respond to nutrient deficiency, and to other biotic and abiotic stresses to survive. It is important to clarify the molecular mechanisms of plant responses to nutrient deficiency, and to utilize the knowledge to improve our life.

In this thesis, I focused on the nutrient deficiency responses in plants using Arabidopsis thaliana. This study consists of two parts: The first part is transcriptome and metabolome analysis of molybdenum (Mo) deficiency and the second part is molecular genetic analysis to identify novel factors regulating plant responses to sulfur (S) deficiency. Mo and S are also important for carbon and nitrogen metabolisms. Nitrogen is a major constituent of proteins, whose deficiency is often a limiting factor for plant growth and productivity.

In the first chapter, I conducted transcriptome and metabolome analysis of plant deficient in Mo. Although Mo is an essential micronutrient in plants, the molecular mechanisms of plant responses to Mo deficiency were not well established. MOT1 is a molybdate transporter belonging to the sulfate transporter family. I described effects of Mo deficiency and defects in MOT1 on plant nitrogen and sulfur metabolisms and transcript accumulations.

In the second and the third chapter, I isolated and analyzed the altered sulfur response (asr) mutants. Forward genetics screens are powerful, unbiased approach to identify novel genes responsible for the biological processes of interest. The asr7-1 showed hypersensitive phenotypes to sulfur deficiency. Genetic analysis identified that ASR7 is CPL1, an RNA polymerase II C-terminal domain phosphatase with two double-stranded RNA-binding domains. Another mutant, asr2-1, showed repressed response to sulfur deficiency. Genetic analysis identified that ASR2 is GLU1, a ferredoxin-dependent glutamate synthase important for photorespiration.

The aims of these two approaches were to clarify the molecular mechanisms of plant responses to nutrient deficiency. From the results, I could propose novel factors and pathways involved in the plant responses to Mo or S deficiency. These findings will be helpful to improve plant productivity and quality.

(1) Effects of molybdenum deficiency and defects in molybdate transporter MOT1 on transcript accumulation and nitrogen/sulfur metabolisms in Arabidopsis thaliana.

Molybdenum (Mo) is a micronutrient essential for plant growth, as several key enzymes of plant metabolic pathways contain Mo cofactor (Moco) in their catalytic centers. Mo-containing oxidoreductases include nitrate reductase, sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. These enzymes are involved in nitrate assimilation, sulfite detoxification, purine metabolism or the synthesis of abscisic acid, auxin and glucosinolates in plants. To understand the effects of Mo deficiency and a mutation in a molybdate transporter MOT1 on nitrogen and sulfur metabolisms in A. thaliana, transcript and metabolite profiling were conducted using the mutant lacking MOT1 in the presence or absence of Mo. Transcriptome analysis revealed that Mo deficiency had impacts on genes involved in metabolisms, transport, stress responses and signal transductions. Transcript level of a nitrate reductase NR1 was highly induced under Mo deficiency in mot1-1. In addition, metabolite profiles were analyzed using gas chromatography time-of-flight mass spectrometry, capillary electrophoresis time-of-flight mass spectrometry and ultra high performance liquid chromatography. The levels of amino acids, sugars, organic acids and purine metabolites changed significantly in the Mo-deficient plants. These results are the first investigation of the global effect of Mo nutrition and MOT1 on plant gene expressions and metabolism.

(2) CPL1 regulates sulfur deficiency response and improves sulfur deficiency tolerance in Arabidopsis thaliana

Sulfur (S) is a macronutrient essential for plant growth and development. To understand the molecular mechanisms of plant adaptation to sulfur deficiency (-S), EMS-mutagenized M2 plants of A. thaliana were screened for mutants with altered patterns of sulfur-responsive gene expression (altered sulfur response; asr mutants). One of the mutants (asr7-1) showed reduced -S-inducible reporter gene expression under both normal and -S conditions. In addition, transcript accumulations of other sulfur-responsive genes such as serine acetyltransferase (SAT1) or a branched-chain aminotransferase (BCAT4) were altered in asr7-1. Growth of asr7-1 under -S conditions was poorer than that of the wild type and -S-responsive amino acid accumulations were altered in the asr7-1, indicating the hypersensitivity of asr7-1 to -S. The asr7-1 mutation was mapped to a 39.2-kb region in chromosome IV, in which only one mutation in nucleotide sequence was found at an exon-intron junction of the CPL1 gene. CPL1 is an RNA polymerase II C-terminal domain phosphatase with two double-stranded RNA-binding domains. We obtained four cpl1 alleles and all of them showed reduced relative growth under -S, indicating that CPL1 is ASR7 and is required for maintenance of growth under -S. In addition, gene expressions and amino acid accumulations were altered in the cpl1 mutants in the same way as asr7-1. Taken together, CPL1 is a novel factor involved in the regulation of sulfur metabolism, gene expression, and tolerance to -S stress.

(3) Ferredoxin-dependent Glutamate Synthase GLU1 Regulates Sulfur Assimilation Pathway in Arabidopsis thaliana

In order to identify the novel regulatory factors of plant gene expressions in response to sulfur deficiency (-S), we took a forward genetics approach using a transgenic A. thaliana line expressing green fluorescent protein (GFP) under the control of a -S-inducible chimeric promoter. EMS-mutagenized M2 plants were screened for mutants with altered patterns of sulfur-responsive gene expressions (altered sulfur response; asr mutants). One of these mutants (asr2-1) showed increased GFP expression under normal condition, and reduced GFP expression under -S conditions. Other sulfur-responsive genes such as adenosine 5'-phosphosulfate reductase (APR1), a sulfate transporter (SULTR4;2) and a serine acetyltransferase (SERAT3;2) were also repressed in asr2-1 under -S conditions. ASR2 was mapped to a 48kb region in chromosome V, and a mutation was found in a ferredoxin-dependent glutamate synthase (GLU1/GLS1/Fd-GOGAT1) gene. GLU1 is important for reassimilation of photorespiratory ammonium into glutamate in plastid. We obtained other glu1 alleles (gls1-30 and gls1-103), and they also showed altered response to -S. These results indicate that ASR2 is GLU1, and GLU1 is involved in sulfur deficiency responses in addition to photorespiration. Increased sulfate accumulations and altered levels of amino acids in the shoots of glu1 under -S conditions were consistent with the altered response to -S. These results demonstrated the involvement of GLU1 in the regulation of sulfur metabolism and sulfur-regulated gene expression.

植物は光合成によって無機物から有機物を生産し、我々動物はそれに依存して生存している。植物の無機栄養獲得と栄養条件に対する応答は植物生産に重要な過程である。本論文は植物の必須元素であるモリブデンと硫黄について、遺伝子転写産物と代謝産物を用いた網羅的解析と、分子遺伝学的、生理学的解析を行ったものであり、序論と3章より成る。

第1章ではシロイヌナズナのモリブデン欠乏とモリブデン酸トランスポーターMOT1の欠損による遺伝子転写産物と硫黄と窒素の代謝に与える影響についての解析を行っている。モリブデン(Mo)は植物の微量必須元素であり、Moを含む補酵素Mocoは硝酸還元酵素や亜硫酸酸化酵素、キサンチン脱水素酵素やアルデヒド酸化酵素などに必要とされる。よってMoの減少は窒素同化や硫黄代謝、プリン代謝や植物ホルモン合成、活性酸素の生成などに影響を与えると考えられる。シロイヌナズナでは硫酸イオントランスポーターに相同性の高いMOT1が根からのMoの吸収に重要であることが最近の研究で明らかにされた。MOT1に変異が入ると植物体のMo濃度が低下し、Mo欠乏の症状が早期に現れる。Mo欠乏が植物の遺伝子発現や代謝産物の蓄積にどのような影響を与えるのかを調べるために、野生型株とMOT1遺伝子のT-DNA挿入破壊株を用いて解析を行った。Moを含む又は含まない固形培地で18日間栽培した植物の葉と根からRNAを抽出してトランスクリプトーム解析を行った。その結果、Mo欠乏によって、窒素、炭素、硫黄の代謝、物質輸送、ストレス応答やシグナル伝達経路などに関わる多くの遺伝子の発現が変化することが示された。特に、硝酸還元酵素NR1の遺伝子転写産物の蓄積がMOT1変異株のMo欠乏条件で強く誘導され、Moによる新たな遺伝子発現制御機構の存在が示唆された。また、根でリン酸や硫酸の輸送に関わる因子の発現が増加しており、硫酸やリン酸の蓄積も変化していたことから、Moの輸送との関連が考えられた。さらに、代謝産物の測定の結果、アミノ酸、糖、有機酸、プリン代謝産物などがMo欠乏の植物で顕著に減少または増加していることが示された。これらの結果は、Mo欠乏とMOT1の変異が植物の遺伝子発現と代謝全体にどのような影響を与えるのかについての初めての知見であり、Mo欠乏が窒素だけでなく、硫黄や炭素、リンなどの代謝に広く関わっていることが示された。

第2章では分子遺伝学的解析によって、CPL1がシロイヌナズナの硫黄欠乏応答を制御し硫黄欠乏耐性を付与する新たな因子であることを発見している。硫黄は植物の生育に必須の元素である。硫黄欠乏に対する植物の応答の分子機構を理解するために、変異原処理をした形質転換シロイヌナズナを用いて、硫黄欠乏に対する遺伝子発現応答に異常の見られる変異株を選抜した。GFP蛍光を指標にマッピングされた範囲の塩基配列を調べた結果、変異は一つであり、原因遺伝子はCPL1と推定された。CPL1はdsRNA binding motifを2つ持つ、転写の調節因子であるCPL1の変異株を独立に2ライン得た。表現形を調べたところ、これらの変異株では生重量の変化が硫黄欠乏に対して感受性が高まっていた。また、硫黄欠乏に対する遺伝子発現の変化が見られた。これらの結果からCPL1が硫黄欠乏に耐性を与え遺伝子発現応答の調節を行う因子であることが示唆され、CPL1は硫黄欠乏などの環境の変化に対して代謝や生育を調整する役割を担っていると考えられた。

第3章:第2章と同様の手法で硫黄欠乏に対する遺伝子発現応答に異常を持つ変異株を選抜しマッピングによって原因遺伝子を解析した結果、フェレドキシン依存型グルタミン酸合成酵素(GLU1)に変異が見つかった。GLU1に変異が入った複数の変異株で、硫酸イオンの蓄積が見られ、GlyやSer、Glnなどのアミノ酸蓄積が硫黄欠乏条件で野生型株よりも顕著に減少し、硫黄欠乏に対する蓄積の増加がみられなくなっていた。これらの結果から、GLU1が硫黄代謝や硫黄応答性の遺伝子発現の制御に関わっていることが新たに示された。

以上本研究は植物のモリブデンおよび硫黄欠乏に対する応答機構について、網羅的解析や分子遺伝学的解析を用いて、新たな現象を見いだし、また、二つの硫黄欠乏に関係する新規遺伝子を単離しその役割を明らかにしたものであり、学術上、応用上貢献するところが少なくない。よって審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。