1

葉緑体ゲノム装置の不連続進化仮説葉緑体ゲノム装置の不連続進化仮説

2

葉緑体ゲノム装置の構造と進化葉緑体ゲノム装置の構造と進化

•葉緑体ゲノムとゲノム装置

•複製酵素，転写酵素， DNA結合タンパク質， RNA結合タンパク質

•比較ゲノム学によるゲノム装置成分の検索

•核様体の比較生化学

•ゲノム装置の不連続進化

3

シアノバクテリアと植物・藻類の葉緑体および

それらの核様体

シアノバクテリアと植物・藻類の葉緑体および

それらの核様体

4

葉緑体ゲノム装置の起源の探索葉緑体ゲノム装置の起源の探索

複製： DNA polymerase(s)

転写：ファージ型 RNA polymerase(s)

DNA 結合タンパク質（転写因子）：HU, DnaB helicaseSiR ( 亜硫酸還元酵素）PEND （包膜の DNA 結合タンパク質）

RNA 結合タンパク質： Rbp / GRP

5

Discontinuous evolution of plastid genomic machinery(1)

Discontinuous evolution of plastid genomic machinery(1)

Adapted from N. Sato (2001) Trends in Plant Science 6: 151-156

DNA polymerase

6

Comparison of various nucleoids

Comparison of various nucleoids

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Lack of prokaryotic DNA-binding proteins in plastids – comparative genomics

Lack of prokaryotic DNA-binding proteins in plastids – comparative genomics

cp, chloroplast genome; nuc, nuclear genome. The Cyanidioschyzon data were used as the ‘nuc’ data for Rhodo- and Chromophytes.

Rhodo-and

Chromo-phytes

Greenalgae

LandplantsProtein

Eu-bacteria

Cyano-bacteria

Buchnerasp.

cp nuc cp nuc cp nuc

HU, IHF 1-4 1 1 0-1 0 0 1 0 0

H-NS,StpA 0-2 0 1 0 0 0 0 0 0

Fis 0-1 0 1 0 0 0 0 0 0

Dps 0-1 1-2 0 0 0 0 0 0 0

DnaB 1 1 1 1 0 0 0 0 0

DnaA 1 1 1 0 0 0 0 0 0

OmpR-like <10 <10 0 1-2 0 0 0 0 0

NtcA 0-1 1 0 1 0 0 0 0 0

CbbR, RbcR 0-2 1-3 0 1 0 0 0 0 0

8

Presence of HU protein in cyanobacteria and rho

dophyte plastids

Presence of HU protein in cyanobacteria and rho

dophyte plastids

9

Sulfite reductase is a major protein in plastid nucleoid

Sulfite reductase is a major protein in plastid nucleoid

10

Formation of particulate complex of purified sulfite reductase with cpDNA

Formation of particulate complex of purified sulfite reductase with cpDNA

Water control BSA control

+ cpDNA (2 hr) + cpDNA (24 hr)

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Models of sulfite reductasesModels of sulfite reductases

E. coli Maize

12

転写活性に対するヘパリンと SiR の効果転写活性に対するヘパリンと SiR の効果

Approximately, 10 sugar residues of heparin counteract with the action of one SiR molecule

13

SiR is also present in isolated moss nucleoidsSiR is also present in isolated moss nucleoids

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Alignment of sulfite reductases of plants and cyanobacteria

Alignment of sulfite reductases of plants and cyanobacteria

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HU と SiR の機能的比較

HU と SiR の機能的比較

HU SiR

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DNA-binding proteins of plastids reported in the literature

DNA-binding proteins of plastids reported in the literature

Protein Plant Target Function Cloned/ Accession

Size (kDa)

Non-specific proteins

HC (plantacyanin) spinach unknown nucleoid structure U76296 10

HlpA Guillardia theta (*1) unknown nucleoid structure AF041468 10

Sulfite reductase pea, tobacco unknown nucleoid structure D83583 68-70

CND41 tobacco unknown repressor/protease D26015 41

Sequence-specific proteins

NdhI (FrxB) Chlamydomonas replication origin replication ? homologue 18

Region D-binding protein spinach region D (psaA promoter) psaA regulation no 31

Region U-binding protein spinach region U (psaA promoter) psaA regulation no 34

PEND pea TAAGAAGT nucleoid anchoring

X98740 130 (70x2?)

PD3 (ENBP1 homologue)

pea AT-rich sequence nucleoid structure?

X98744 170/130/63

CDF2 spinach AAGAGGCTCGTGGG rrn regulation no 33/35

CDF1 pea intergenic rbcL-atpB transcription ? no 115

AGF barley AAG box psbD activation no unknown

PGTF barley PGT(plastid GT) box psbD activation no unknown

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紅藻と植物の DNA polymerases紅藻と植物の DNA polymerases

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植物オルガネラ型 DNA polymerases の系統植物オルガネラ型 DNA polymerases の系統

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葉緑体とミトコンドリアの複製系の起源葉緑体とミトコンドリアの複製系の起源

植物・藻類では，葉緑体とミトコンドリアの複製系は極めて似ており，同一の DNA polymerase が働いている。

しかしこの DNA polymerase の起源は，シアノバクテリアや α プロテオバクテリアに求めることはできない。

動物や菌類と植物・藻類では，ミトコンドリアDNA polymerase が異なっている。

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Origin of NEP

Recent evolution of T7-like RNA polymerases

Origin of NEP

Recent evolution of T7-like RNA polymerases

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Origin of NEPOrigin of NEP

In angiosperm chloroplasts, two types of RNA polymerases (RNAP) are present: one is a prokaryotic RNAP called PEP, which is encoded in the chloroplast genome, while another is a phage-type RNAP called NEP, which is encoded in the nuclear genome.

The phage-type RNAP consists of a single polypeptide, and functions in mitochondria of most eukaryotes including yeast and human.

We analyzed the phage-type RNAP in the moss Physcomitrella patens.

22

Model of organellar RNA polymerases in in higher plants

Model of organellar RNA polymerases in in higher plants

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Two cDNAs: RpoT1 and RpoT2 Two cDNAs: RpoT1 and RpoT2

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Expression and purification of PpRPOT proteins

Expression and purification of PpRPOT proteins

Nuclear- encoded phage-type RNA polymerase of Physcomitrella patens (moss)

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60 ng/l

2 g

30 ng/ l

80 ng/l

40 ng/ l

Enzymatic activity of the T7-type

RNA polymerases

in Physcomitrella

Enzymatic activity of the T7-type

RNA polymerases

in Physcomitrella

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Mitochondrial targeting of the PpRPOT proteins

Mitochondrial targeting of the PpRPOT proteins

27

Phylogeny of T7-type RNA

polymerases

Phylogeny of T7-type RNA

polymerases

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Phylogeny of T7-type RNA polymerases(2)

Phylogeny of T7-type RNA polymerases(2)

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Residues750-776 M F L G Q F R L Q P T I N T N K D S E I D A H K Q E S

S. cerevisiae 1129-1155Q V E T N L Q T V F I S D P F A V N P V N A R R Q K AD. discoideum 822-849N I R T L E C D F I V V H N D D L L Q V D S N R Q R S

G. theta S V E T A V Q N I S L L K C D E N G P I N K L K Q R TP. provasolii K I N T V L Q T I S V D I H S E A L P V V S A K Q R S

PpRPOT1 960-986L V K T S L Q V L A L R N L D A D Q P V L V Q R Q K SPpRPOT2 938-964L V K T S L Q V L A L R N T D D N H P V L A S R Q R S

A. capillus-veneris Q V R T S L Q I L A L T D S N D T N M I M V R R Q K S

P. taeda L V R T S P Q I W A L R D E T X K V W A I H K K L H S

A. thaliana 851-875L V K T T L Q V L T L S - - R E T D K V M A R R Q M TN. sylvestris 876-901L I K T S L Q I L T L Q - - R E T D K V M V K R Q E TC. album 863-877L V K T S L Q V L T L R - - C D T D K V M A K R Q R TZ. mays 850-874L I K T S L Q V L T L Q - - R E T D K V M V K R Q K TT. aestivum 880-904L I K T S L Q V L T L Q - - R E T D K V M V K R Q R T

N. sylvestris 895-920L I K T S L Q I L T L Q - - R E T E K V M V K R Q R TA. thaliana 886-910L V K T S L Q T L S L Q - - H E T D Q V I V R R Q R T

A. thaliana 868-892L I R T S L Q V L A L Q - - R E G N T V D V R K Q R TZ. mays 827-851M I R T S L Q C L A L R - - R E G D A I A I Q R Q K AT. aestivum 824-848M I R T S L Q C L A L R - - R E G D A I A T Q R Q K A

Species SequenceBacteriophage T7Protist

Algae

Moss

Fern

Gymnosperm

AngiospermsMitochondrion-type

Mt/Pt-type

Plastid-type

Signature sequenceSignature sequence

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NEP-dependent promoters of

chloroplast genes might be specific to

angiosperms

NEP-dependent promoters of

chloroplast genes might be specific to

angiosperms

NCII : Non-consensus type promoter II, which is transcribed by NEP.

31

Recent origin of NEP (2)Recent origin of NEP (2)

The NEP, a nuclear-encoded phage-type RNA polymerase of chloroplasts, has been created by duplication of the gene encoding a mitochondrial counterpart.

Phylogenetic analysis of the polymerases as well as the structure of NEP-dependent promoters suggest that this gene duplication occurred after the evolution of angiosperms.

Plant & Cell Physiology 43: 245-255 (2002)

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Conflicting results on the targeting of the two RNAPs of Physcomitrella paten

s

Conflicting results on the targeting of the two RNAPs of Physcomitrella paten

s

1. Kabeya and Sato (2002) Plant Cell Physiol. 43: 245-255

Targeting to mitochondria (no targeting to chloroplasts)

2. Richter et al. (2002) Gene 290: 95-105

Dual targeting (mostly to chloroplasts)

33

Chloroplast targeting ??Chloroplast targeting ??

Forced translation from the first AUG

34

Mitochondrial targeting of the PpRPOT proteins (2)

Mitochondrial targeting of the PpRPOT proteins (2)

Translation within the natural context

35

Two methionine codonsTwo methionine codons

36

GFP in transient expressionGFP in transient expression

37

Stable transformantsStable transformants

38

ImmunoblotImmunoblot

39

Tagetitoxin sensitivityTagetitoxin sensitivity

40

Translation efficiencyTranslation efficiency

41

コケでも被子植物でもコケでも被子植物でも

これまで他のグループによって２重ターゲティングが提唱されていた，細胞核にコードされた RNA polymerase (RPOT) は，いずれもミトコンドリアだけにターゲティングされること，その理由は，本来の 5’ UTR コンテキストでは，２番目のメチオニンコドンだけからしか翻訳されないためであること，が判明しいた。

従って，本来の 5’ UTR を持たない人工的に作られた GFP融合タンパク質に基づくターゲティングは，正しい結果をもたらさないということが教訓である。

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Dually targeted DNA-binding protein, PENDDually targeted DNA-binding protein, PEND

43

若いエンドウの葉では，核様体が包膜に結合している

若いエンドウの葉では，核様体が包膜に結合している

播種後６日目

播種後１４日目

5 µm

44

核様体の包膜結合に関与する DNA 結合タンパク質PEND protein: Sato et al. (1993) EMBO J. 12: 555-561.核様体の包膜結合に関与する DNA 結合タンパク質

PEND protein: Sato et al. (1993) EMBO J. 12: 555-561.

45

Import of the PEND proteinImport of the PEND protein

46

Import of the PEND protein (2)Import of the PEND protein (2)

47

Import of the PEND protein (3)Import of the PEND protein (3)

Localization of the full-length protein to the chloroplast envelope

48

Dual targeting of the PEND proteinDual targeting of the PEND protein

Initial translation product is targeted to plastid envelope.

The N-terminus is processed. The C-terminus is involved in membrane-binding.

If the N-terminal half of the mature PEND protein is cleaved, this polypeptide may be re-targeted to the nucleus.

BnGSBF1, a PEND homolog, is supposed to act as a transcription regulator in CAB gene.

49

Selection of binding sites for the PEND protein (cbZIP region)

Selection of binding sites for the PEND protein (cbZIP region)

50

Binding sites for the PEND protein in the pea cpDNA

Binding sites for the PEND protein in the pea cpDNA

51

Gel-mobility shift assay of DNA-binding specificity of the cbZIP domain

Gel-mobility shift assay of DNA-binding specificity of the cbZIP domain

52

53

54

55

Discontinuous evolution of plastid genomic machinery(1)

Discontinuous evolution of plastid genomic machinery(1)

Adapted from N. Sato (2001) Trends in Plant Science 6: 151-156

DNA polymerase

56

Phase 1: 複製系の入れ換えと転写因子の喪失Phase 1: 複製系の入れ換えと転写因子の喪失

57

Phases 2-3: 真核型転写因子と転写系の移入Phases 2-3: 真核型転写因子と転写系の移入

58

プラスチドは，現存するシアノバクテリアの共通の祖先に近い原核生物が，ミトコンドリアをもつ真核細胞内に共生することによって生じたが，シアノバクテリアの系統におけるプラスチドの位置や最も起源に近いプラスチドについては，まだ研究が必要である。

緑色系統と紅藻系統，紅藻とヌクレオモルフの単系統性を支持するゲノムデータが示された。

ゲノム全体の比較により，シアノバクテリアからプラスチドにいたる系統と進化を跡づけることができ，これによって，新規光合成関連遺伝子の同定が進められている。

ゲノム装置（核様体）の構成成分では，シアノバクテリアが持っているものの大部分がプラスチドでは失われていること，紅色系統と緑色系統では成分が異なること，また，高等植物では真核細胞由来の新たな成分が付け加わっていること，などが判明した。

まとめまとめ