Abstract

　Stem cells could be defined as cells having ability to replenish themselves through self-renewal as well as the potential to generate differentiated cells. Recent advance on analysis of stem cells revealed common gene sets as well as regulatory mechanisms underlying stem cell regulation in plants and animals. However, most plant cells are considered to have totipotency, and thus it is difficult to precisely define stem cells in plants. It may be suitable to understand stem cell in plants as a transient cellular state to achieve activity to act as stem cells. In plants, specialized tissues containing undifferentiated stem cells are commonly referred to as meristems. Analysis on molecular mechanisms regulating development and maintenance of meristems advanced our understanding of regulatory mechanisms underlying. However, our current knowledge is mainly limited to factors involved in transcriptional regulation, and regulatory mechanisms directly involved in activity of cell metabolism that may affect meristematic activity remain to be elucidated. In order to identify genes involved in regulating meristematic activity of vascular cambium, attempts using the gene trap strategy were performed to obtain procambium genes.

　The procambium stage of vascular development, which precedes the specification of each mature vascular cell, remains relatively uncharacterized. Anatomical analysis of early vascular development including procambium development has been done extensively. The primary vascular tissues are produced from procambial initials in the apical meristems of shoots and roots. Cytologically, procambium cells become apparent as cytoplasm-dense, elongate, and narrow cells, with the long axis parallel to the axis of the procambium strand. Although these morphological characteristics are useful for recognizing procambium cells from ground cells, they do not provide the developmental cue of the procambium. In addition, how cell fate is specified to vascular development, which precedes morphological changes, is unknown. The gene trap strategy aims to visualize expression patterns and functional domains of random genes in the genome by tagging endogenous genes to produce chimeric proteins with endogenous proteins and reporter proteins. The strategy is quite useful for identifying specific genes expressed temporally and/or spatially. Using this strategy, five gene trap lines showing β-glucuronidase (GUS) staining in procambium cells were isolated and the causal genes for this staining in four of the five lines were identified. Procambium develops through undifferentiated meristematic, preprocambium, and procambium stages. The specificity of staining pattern of those procambium staining lines have varieties, and it could be subdivided into two groups (Figure 1). (1) KG09306, KG12346 and KG12419 showed GUS staining from undifferentiated GM cells near elongating higher order vein in leaves. In contrast, (2) KG11997 and KG14159 were GUS stained from fully elongated procambium cells. From these results, the PINHEAD gene (KG09306), the Gamma-Glutamyl Hydrolase 1 (GGH1) gene (KG12346) and the KATANIN gene (KG12419) are suggested to be expressed through the three stages, whereas the At3g09070 gene (KG11997) and the causal gene of KG14159 are expressed from the procambium stage. Thus, these genes are useful for future analysis as markers for different stages of procambium development.

　Next, the GGH1 gene, which was tagged by reporter gene in KG12346, and orthologous GGH2 and GGH3 were characterized in detail. GGH cleaves the polyglutamate chain of folates. Folates are essential cofactors involved in one-carbon transfer reactions for amino acids and nucleic acid metabolism and metabolic regulation. Synthesis of purines, thymidylate, pantothenate, and formylmethionyl-tRNA, as well as interconversion of glycine and serine is mediated by tetrahydrofolate and its derivatives. Folates are also involved in DNA methylation reactions crucial for proper regulation of gene expression. Thus, appropriate control of activity of folates is inevitable for all organisms. Like in other organisms, plant folates exist as polyglutamyl form, having γ-liked chain of glutamyl residues attached to the first glutamate. Anionic nature appended by glutamate residues increases binding ability of folates to folate binding proteins. Thus, proper control of glutamate chain lengths of folates should be achieved in cells. GGH1 and GGH2 were expressed in cells having meristematic activity (Figure 2A - D). Overexpression of each three GGH genes, tandemly placed in Arabidopsis genome, caused similar developmental abnormalities indicating reduction of meristematic activity (fusion of cotyledons or adult leaves, reduction of meristematic cells in SAM; Figure 2E - G). Conversely, suppression of all GGH genes by RNAi caused developmental abnormalities indicating enhancement of meristematic activity (overproduction of floral organs, ectopic formation of bracts, ectopic secondary lamina formation; Figure 2H - J). These results indicate that GGH is a negative regulator of meristematic activity expressed in meristematic tissues. Thus, I propose a novel molecular mechanism regulating meristematic activity via controlling glutamate chain lengths of folates. In this model, by catalyzing cleavage of glutamate residue of poly-glutamated folates that is essential to retain meristematic activity, GGH induce meristematic cells to differentiate into mature tissues (Figure 3). Although the biological significance of folates is obvious, the novelty of this model is suggesting significance of controlling glutamate chain lengths of folates. The fact that poly-glutamated folates are preferred form for most enzymes supports the model.

　To elucidate the significance of controlling glutamate chain length of folates in plant development, effects of folate biosynthesis inhibitors and folates with different lengths of glutamate chain on plant development were examined. Consumption of the SAM region and reduction of root length were observed in plants treated with folate biosynthesis inhibitor. Both defects could be overcome by supplying pteroyl pentaglutamate but could not by supplying pteroyl monoglutamete (Figure 4A,B). These results indicate the role of poly-glutamated folate in maintaining proliferative activity of meristematic cells in both root and shoot meristematic region. Then, to elucidate the molecular basis of developmental defects of folate biosynthesis inhibitor treated plants, expression patterns of several genes implicated in development or maintenance of SAM were examined. Folate biosynthesis inhibitor treatment to plants induced down-regulation of genes involved in SAM development and/or maintenance. On the contrary, inhibitor treatment induced ectopic expression of genes act as negative regulators of meristematic activity and a gene related to xylem differentiation. The effect of folate supplementation on in vitro tracheary element (TE) differentiation system was examined subsequently. Pteroyl penta- glutamates inhibited TE differentiation of cultured Zinnia cells, while mono- glutamated folates did not have inhibitory effect (Figure 4C). In TE differentiation inhibited cells by pteroyl pentaglutamate treatment, expression of marker genes up-regulated in late stage1 and early stage 2 were sustained and expression of marker genes up-regulated in stage 2 were delayed (Figure 4D). Thus, TE differentiation inhibited cells were seem to be sustained in the late stage 1 or early stage 2, when cells are about to acquire identity as meristematic procambium-like cells. These results indicate that poly-glutamated folates are inevitable for plants to retain meristematic activity, and match well with the proposed model.

figure 1. Gene trap lines exhibiting GUS staining in the procambium

GUS staining observed near the terminal site of elongating procambium tissues of higher order vein in young leaves of KG09306 (A), KG12346 (B), KG12419 (C), KG11997 (D) and KG14159 (E). Images are DIC images. To facilitate distinguishing GUS stained cells, color of images are reversed. GUS stains are observed as red staining. Isodiametric, polygonal GM cells are blue outlined, unelongated preprocambium cells are red outlined and elongated procambium cells are yellow outlined. Bars: 20 μm

Figure 2. Analysis of GGH genes in Arabidopsis

(A - C) GUS staining pattern of stage 18 embryo of KG12346 plant (A), inflorescence of pGGH1::GUS plant (B) and inflorescence of pGGH2::GUS plant (C).

(D) In situ localization of GGH1 mRNA in wild-type Arabidopsis plants. GGH1 is expressed in SAM (blue arrow) and young floral organs (red arrow).

(E) Fused cotyledons of 35S::GGH1 plant.

(F) Fused rosette leaves of 35S::GGH1 plant.

(G) Complete loss of SAM observed in 35S::GGH1 plant.

(H) Flower of GGH RNAi plant with 6 petals.

(I) Flower of GGH RNAi plant with ectopic bract (red arrow).

(J) Cauline leaf of GGH RNAi plant with ectopic secondary lamina (red arrow).

Figure 3. A model of folate-mediated regulation of meristematic activity by GGH

Figure 4. Physiological analysis of differential activities of mono- or penta- glutamated folates

(A) Effect of sulfanilamide treatment on SAM and recovery by penta-glutamated folates.

(B) Effect of sulfanilamide treatment on root lengths and recovery by penta-glutamated folates.

(C) Effect of folate treatment on in vitro TE differentiation.

(D) Effect of folate treatment on expression pattern of developmental stage1 marker gene ZePR.

Sulf = sulfanilamide, Glu = pteroyl pentaglutamate, FA = pteroyl monoglutamate, (1) =1 μM, (50) = 50 μM

　本論文は、植物における幹細胞の維持と分化に関して、分子遺伝学的、植物生理学的に解析したものであり、6章からなる。第1章では、Prefaceとして、この研究の背景と研究を始めるにあたっての動機を述べている。第2章では本研究で使われた材料と方法について記述されている。第3、4、5章は研究の結果とその考察であり、第3章では、ジーントラップラインを用いた形成層特異的な遺伝子の同定について、第4章では、得られた形成層特異的遺伝子のうち、ポリグルタミン酸型葉酸からグルタミン酸鎖を切断する酵素をコードするGGH1とその類似遺伝子の分子遺伝学を用いた機能解析について、第5章では、ポリグルタミン酸型葉酸の生理機能について述べられている。第6章では得られた結果を受けて、総合的にポリグルタミン酸型葉酸の幹細胞の維持に関する役割について考察している。

　植物メリステムは、新たな多様な細胞を作り出していく分裂組織で、そこには幹細胞が存在し、その維持と多様な細胞への分化が厳密にコントロールされている。論文提出者は、植物メリステムにおける幹細胞の維持と分化の制御機構を知る目的で、この制御に関与する因子の単離とその機能解析を行った。

　論文提出者は、まず、シロイヌナズナジーントラップラインのスクリーニングにより、前形成層に強く発現するラインの探索を行った。そして、26000のラインのから5ラインの前形成層特異的ラインの同定に成功した。そして、その遺伝子発現の詳細な解析から未分化状態のground meristem細胞(GM細胞)から発現するライン(KG09306、KG12346、KG12419)と伸長した前形成層細胞に特徴的に発現するライン(KG11997、KG14159)の分離に成功し、維管束のごく初期段階を区別できるマーカーの獲得に成功した。また、その挿入タグを手掛かりに原因遺伝子の同定を行い、4ラインの原因遺伝子(KG09306:PINHEAD、KG12346:Atlg78660、KG12419:AtKTN，KG11997:At3g09070)の同定に成功した。これらの遺伝子は、これまでほとんど明らかになっていない、維管束幹細胞の形成の初期過程を明らかにするtoolになるとして、高く評価された。

　次に、論文提出者は、KG12346の原因遺伝子の発現・解析を進めた。KG12346はgamma-glutamyl hydrolase 1 (GGH1)をコードするAtlg78660遺伝子内にタグが挿入されていた。この酵素は葉酸のグルタミン酸鎖切断能を持つことが報告されている。しかし、この酵素と形態形成との関連はまったく明らかになっていなかった。GGH1遺伝子はシロイヌナズナゲノム中に他の2つのホモログ遺伝子(GGH2、GGH3)と一緒にタンデムに並んで存在していた。そこで、論文提出者は3つのGGH遺伝子の発現パターンをGUS発現とin situハイブリダイゼーションにより解析した。その結果、2つの遺伝子が前形成層、茎頂分裂組織周辺及び若い花器官など、メリステムで強く発現していることを明らかにした。さらに、その機能解明のために各GGH遺伝子の過剰発現体を作成したところ、いずれのGGH遺伝子導入でも子葉やロゼット葉の融合、頂芽優勢の弱化、茎頂分裂組織の縮小が観察され、茎頂部におけるメリステム活性が低下していることが示された。逆に、GGH遺伝子全ての発現を抑制しうるRNAiコンストラクトを作成し、シロイヌナズナ植物体に導入した。このうち、全てのGGH遺伝子発現の低下した植物体では、花器官数の増加、花器官への異所的な苞葉形成、葉身上への異所的な二次葉身の形成が観察され、メリステム活性の亢進を示した。これらの結果を総合して、論文提出者は、GGHタンパク質は植物体の前形成層のみならず茎頂をも含むメリステム領域において、メリステム活性を負に制御していると結論づけた。本結果は、葉酸の代謝酵素がメリステムの活性を負に制御することを初めて見いだしたもので、高く評価される。

　さらに、論文提出者はグルタミン酸型葉酸の機能を詳細に調べるために、生理学的な解析を行った。まず、葉酸生合成の拮抗的阻害剤であるsulfanilamideを用いて、メリステム形成における葉酸の必要性を検討した。その結果、葉酸生合成阻害により、茎頂分裂領域の縮小や根の伸長阻害が引き起こされ、単グルタミン酸型葉酸添加によっては回復しないが、5グルタミン酸型葉酸の添加により回復することを見いだした。この結果は、多グルタミン型葉酸が根端・茎頂のメリステム活性の維持に必須であることを初めて明らかにしたものと評価される。次に、多グルタミン型葉酸が維管束メリステムにも機能を持つかどうかを、ヒャクニチソウ管状要素分化系を用いて解析した。その結果、3から6のグルタミン酸鎖を持つ葉酸を添加したときに、管状要素分化が阻害されることがわかった。一方で、単グルタミン酸型の葉酸は分化に影響を与えなかった。分化のマーカー遺伝子の発現解析の結果、多グルタミン酸型葉酸は未分化状態の細胞から前形成層様細胞への分化の過程を阻害していることを見いだした。この結果は、多グルタミン酸型葉酸が植物におけるメリステムすべてにおいて、その幹細胞の維持に関与していることを示した、世界初の研究として高く評価された。

　なお、本論文第1章は澤進一郎、加藤友彦、佐藤修正、田畑哲之、福田裕穂氏との共同研究であるが、論文提出者が主体となって解析を行ったもので、論文提出者の寄与が十分であると判断する。

　以上、ここに得られた結果の多くは新知見であり、いずれもこの分野の研究の進展に重要な示唆を与えるものであり、かつ本人が自立して研究活動を行うのに十分な高度の研究能力と学識を有することを示すものである。よって、名川信吾提出の論文は博士(理学)の学位論文として合格と認める。