細胞生物學 : 葉綠體 粒線體 12/5/16 葉綠體與粒線體的生理功能 謝明勳副研究員 中央研究院植物暨微生物學研究所. Endosymbiosis and The Origin of Eukaryotes

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1 細胞生物學 : 葉綠體 粒線體 葉綠體與粒線體的生理功能 葉綠體 光合作用 謝明勳副研究員 中央研究院植物暨微生物學研究所 粒線體 什麼生物同時具有葉綠體與粒線體? 呼吸作用 真核生物的起源 真核生物的起源 Endosymbiosis and The Origin of Eukaryotes 1.2 billion years ago Endosymbiosis theory 內共生假說 The mitochondria ( 粒線體 ) of eukaryotes evolved from α-proteobacteria (α- 變形菌 ) living within their host cell. The chloroplasts ( 葉綠體 ) of eukaryotes evolved from endosymbiotic cyanobacteria ( 藍綠菌 ). 1.5 billion years ago 阿拉伯芥 (Arabidopsis thaliana) : 當代分子遺傳研究最受歡迎的模式植物 Transmission electron micrograph of a cell in an Arabidopsis leaf - 生活周期短 : 約 6-8 周 - 植物體小 : 成株約 30 公分高 - 種子產量高 : 每棵植物可產生數千顆種子 - 基因體小 :5 對染色體, 約 1.25 x 10 8 鹼基對 (base pairs, bp) - 基因體已定序完成 : 約 3 萬個基因 - 知識 技術發展迅速 : 研究者眾 - 大量的突變種可供研究 : 阿拉伯芥種源中心 1

2 Transmission electron micrograph of a chloroplast in an Arabidopsis leaf cell Thylakoids The light reactions and chemiosmosis: the organization of the thylakoid membrane 光反應 Stroma thylakoids Granal thylakoids Stroma The transfer of electrons and protons in the thylakoid membrane is carried out vectorially by four protein complexes 碳反應 ( 暗反應 ) The Calvin cycle Rubisco Rubisco: Ribulose-1,5- bisphosphate carboxylase oxygenase 植物葉片中含量最豐富的蛋白質, 也可能是地球上含量最多的蛋白質 Rubisco: 卡爾文循環的第一個酵素 Carboxylation Rubisco catalyzes both carboxylation and oxygenation reactions Rubisco active site Oxygenation Ribulose 1,5-bisphosphate carboxylase / oxygenase (Rubisco) catalyzes the reaction between Ribulose 1,5- bisphosphate (RuBP) and CO 2 or O 2 Therefore it functions as either a carboxylase or an oxygenase Carboxylation produces two molecules of 3-phosphoglycerate (3-PGA) Oxygenation produces one 3-PGA and one 2-phosphogylcolate (2-PG) Rubisco: Ribulose-1,5-bisphosphate carboxylase oxygenase Recycling the wasteful oxygenation product 2-PG costs energy and releases fixed CO 2 Tabita, F.R., Satagopan, S., Hanson, T.E., Kreel, N.E. and Scott, S.S. (2008). Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships. J. Exp. Bot. 59: by permission of Oxford University Press. 2

3 澱粉與蔗糖的合成 粒線體 (mitochondrion) 的構造 澱粉 : 貯藏 蔗糖 : 運送 粒線體皺褶 粒線體基質 粒線體外膜 粒線體內膜 複數 : mitochondria 細胞內的呼吸作用 Glycine Cysteine O-Acetylserine Serine Asparagine Glucose Fructose 1,6-BP 3-Phosphoglycerate Phosphoenolpyruvate Pyruvate Acetyl-CoA Tryptophan Tyrosine Phenylalanine Anthranilate Chorismate Erythrose 4-phosphate DAHP Shikimate Alanine Prephenate 糖酵解克勃氏循環氧化磷酸化 Aspartate Oxaloacetate Citrate Aspartate 4-phosphate Aspartate 4-semialdehyde Homoserine 2,3-Dihydro- dipicolinate Malate Fumarate Isocitrate Oxalosuccinate Homoserine Succinate α-ketoglutarate 4-phosphate Cysteine DAP Threonine Cystathionine Pyruvate Pyruvate α-ketobutyrate Homocystine Leucine Valine Isoleucine Methionine Lysine Proline Glutamate Ornithine Glutamine Citrulline Histidine Arginine Chemiosmosis couples the electron transport chain to ATP synthesis Electron transport chain - oxidative phosphorylation kda Polypeptides Complex I NADH dehydrogenase (7) Complex II Succinate dehydrogenase Complex III Cytochrome C oxidoreductase (1) Complex IV Cytochrome C oxidase (3) Complex V ATP synthase (2) 3

4 Development of a chloroplast from a proplastid Chloroplast biogenesis Stages in the development of chloroplasts 與葉綠體功能有關的阿拉伯芥突變種 in an Arabidopsis leaf 3a60 3a87 3s533 u866 3a130 3a300 3s593 3s874 3a303 5s11 3s231 5s48 3s523 6a27 3a234 u73 6a27 3a nm 4

5 U866 Arabidopsis isph-1 albino mutant Ming-Hsiun Hsieh, and Howard M. Goodman Plant Physiol. 2005;138: by American Society of Plant Biologists The development of chloroplasts is arrested at an early developmental stage in an albino mutant WT albino mutant Chloroplast division Hsieh et al., 2008 Electron micrograph of dividing plastids Position of the FtsZ ring during sequential stages of chloroplast division The position of the FtsZ ring is indicated by arrows. FtsZ proteins: related to the cytoskeletal component tubulin 5

6 Single giant chloroplast in Arabidopsis ftsz mutant WT ftsz mutant FtsZ is dispensable for plant development Chloroplast movement KO: knock-out Chloroplast distribution in photosynthesizing cells of the duck weed Lemna accumulation response avoidance response Arabidopsis NPL1: A Phototropin Homolog Controlling the Chloroplast High-Light Avoidance Response Takatoshi Kagawa, Tatsuya Sakai, Noriyuki Suetsugu, Kazusato Oikawa, Sumie Ishiguro, Tomohiko Kato, Satoshi Tabata, Kiyotaka Okada, Masamitsu Wada4 6

7 Chloroplast movement Photoperception Phototropin a blue light receptor Chloroplast genome Signal transduction? Chloroplast movement Actin filaments 葉綠體與粒線體都有自己的 DNA DNA in chloroplasts Chloroplast gene expression machinery Replication of plastid DNA - Plastid DNA replication is not linked to organelle division. Replication Chloroplast DNA Transcription 轉錄 Chloroplast mrna 複製 - The number of genome copies present in plastids can vary during development. ~40 copies in shoot meristem cells, increases to ~600 copies in leaf primordia, decreases again in mature leaves Translation 轉譯 Chloroplast Protein 葉綠體與粒線體內的基因表現機制, 比較類似原核生物 -- 2 origins of replication (oria and orib) -- Displacement loop (D-loop): indicative of an origin of replication 7

8 葉綠體 DNA 的複製 A chloroplast from a leaf of timothy grass (Phleum pratense) -Chloroplast DNA replication is not linked to organelle division. 葉綠體的分裂與細胞分裂無關 葉綠體 DNA 的複製與葉綠體的分列無關 - The number of genome copies present in chloroplast can vary during development. 葉綠體 DNA 的套數從幾十到幾百都有 plastoglobulus (pl. plastoglobuli): A densely staining droplet found, often in large numbers, in plastids of plant cells. A chloroplast from a leaf of timothy grass (Phleum pratense) 以阿拉伯芥為例, 請問植物的葉綠體內約有多少個基因與蛋白質? 請問植物的粒線體內約有多少個基因與蛋白質? Ribosome ( 核醣體 ) 以阿拉伯芥為例, 請問植物的葉綠體內約有多少個基因與蛋白質? 87 基因, >3,000 蛋白質請問植物的粒線體內約有多少個基因與蛋白質? 57 基因, 2,000 ~ 3,000 蛋白質 Mitochondria and chloroplasts are semiautonomous organelles 阿拉伯芥的細胞核基因體 :125 Mb; ~30,000 個基因 阿拉伯芥的葉綠體內約有 3,500 種蛋白質阿拉伯芥的葉綠體基因體 (154 kb):~87 個基因 阿拉伯芥的粒線體內約有 2,500 種蛋白質阿拉伯芥的粒線體基因體 (367 kb):~57 個基因 8

9 Gene content of tobacco plastid DNA The transfer of electrons and protons in the thylakoid membrane is carried out vectorially by four protein complexes Plastid genome: Plastome psb pet psa ndh atp rpl, rps, ycf, orf 植物葉綠體的基因體大約有 個基因 每個植物細胞大約有 100 個葉綠體 什麼基因還留在葉綠體內? I. Genes involved in plastid gene expression - rrnas, trnas - ribosomal proteins - RNA polymerase II. Genes involved in photosynthesis (electron transport chain) - 28 thylakoid proteins psa and psb subunits - ATP synthase subunits (atp) - NADH dehydrogenase subunits (nad) - Cytochrome b6f subunits (pet) III. RUBISCO large subunit (rbcl) Organized in operons; some gene orders conserved with bacteria 每個葉綠體大約有 100 個基因體 ; 每個葉綠體的基因體大約有 100 個基因 多數源自藍綠菌的基因已轉移到細胞核 Evolutionary trend towards reducing organelle gene content Genome Rikettsia prowazekii (alpha proteobacterium) Reclinomonas americana (protozoan) mitochondria Marchantia polymorpha (liverwort) mitochondria Arabidopsis thaliana mitochondria Homo sapiens mitochondria Protein coding genes Leister (2003) Similar trend in the chloroplasts 在葉綠體演化過程中, 大部份原來屬於藍綠菌的基因, 已經轉移到細胞核裡 9

10 Human Mitochondrial Genome 人類粒線體的基因體全長 16.5 kb, 只有 13 個可以轉譯成蛋白質的基因 The genome of human mitochondria contains 16,569 base pairs of DNA organized in a closed circle. These encode: 2 ribosomal RNA (rrna) molecules 22 transfer RNA (trna) molecules 13 polypeptides 7 subunits that make up the mitochondrial NADH dehydrogenase 3 subunits of cytochrome c oxidase 2 subunits of ATP synthase cytochrome b 絕大部分的粒線體蛋白質, 都是在細胞質內合成, 然後運送到粒線體內 Electron transport chain - oxidative phosphorylation kda Polypeptides Complex I NADH dehydrogenase (7) Complex II Succinate dehydrogenase Complex III Cytochrome C oxidoreductase (1) Complex IV Cytochrome C oxidase (3) Complex V ATP synthase (2) Vegetative segregation can lead to variegation 綠色 : 正常的葉綠體白色 : 突變的葉綠體 10