Genome of plastids and mitochondria

General features of P & M

• Double membrane

• Multiplication/reproduction by division

• Their own DNA and ribosomes (70S)– Synthesis of only small portion of proteins– Transfer of genes to nuclear genome

• Endosymbiotic origin

• Role in energetic metabolism

Origin of plastids and mitochondria

Gillham 1994 *The tree of Eucaryots has been updated!

Primary, secondary, tertiary endosymbiosis

– varying number of membranes (alt. nucleomorph)

Primary (1,6 BY) - Glaucophytes- Rhodophytes- Chlorophytes

Secondary - Euglenophyta - Chlorarachniophyta - Chromalveolata

Glaucophytes

Transient endosymbiosis(sea snail Elysia chlorotica)

- active chloroplasts of sea weed Vaucheria

- active for up to eight months

- PsbO (MSP) present in Elysia genome!!!

(Rumpho et al. 2008 PNAS)

Rumpho M. et al., 2000

Stromules of chloroplasts (tubular structures) - contraversion if they allow exchange of genetic material and proteins

- clear communication with other organeles (ER, mitochondria?)

Schattat M et al. Plant Cell 2012;24:1465-1477

Hanson M, Sattarzadeh A. Plant Cell 2013;25:2774-2782: … transfer YES

transfer NO

Reproduction of plastids

Lopez-Juez E., 2007

Combination of procaryoticFtsZ a eucaryotic dynamine circle

Internal circle:1. FtsZ 2. FtsZ + dynamin3. dynamin

external: dynamin (role of ER?)

MITOCHONDRIA: FtsZ missing in plants, other factors, role of ER

Plastome a chondriomeversus nuclear genome

Gene functions in plastome (and chondriome)

+ genes for rRNA and tRNA (some are missing in mitochondria)

Nuclear encoded proteins in P&M(predikce Target P u Arabidopsis – Emanuelsson et al., J Mol Biol)

Mitochondria ~ 10 % (cca 2500 genes)

Plastids ~ 14 % (cca 3500 genes)

Approx. 1/4 genes necessary for P&M

- many proteins do not originate from the endosymbiont, but host genome or other endosymbiont

- many genes/proteins originating from endosymbionts used for various functions in cytoplasm

Leister D., TRENDS in Genetics 19: 47, 2003.

Fate of genesfrom theendosymbiont

Arabidopsis

nucleome: approx. 26.000 genesplastome: 87

proteins in plastid: ~ 3500 genes

endosymbiont (~ 4500 genes)(blue-green algae:

~ 3000 – 7000 genes)

- more complex regulation of gene expression- use of genes/proteins for secondary functions- teoreticalla higher mutation speed in organells

- no recombination (correction of mutations) in sexual reproduction- however, chloroplast genes highly conserved!

Causes of gene transfer to nucleus

Protein products have to be imported back to the organelle!

Inaba and Schnell; Biochem J (2008)

Major pathway:TOC-TIC translocons (channels + chaperons!)

Transite peptide: 30-100 amk (2-4.000 proteins),

mitochondria: TOM-TIM

Transport of proteins to plastids

OM – outer membrane

ER-CP – glycoproteins

Uncleaved TP

Transport to thylakoids

Lumen: prokaryotic transport systems Thylakoid membrane – spontaneous incorporation (+ plastid encoded

proteins)

Recent gene transfer to nucleus

(if the gene obtains transite peptide, it can be lost from the organelle)

• Fusion with transite peptide of an imported protein

• e.g. mitochondrial Rps11 paralogs in rice use TP of Cox and mitochondrial ATPase subunit

(residues of mt gene are still transcribed)

Kadowaki et al., EMBO J. 1996

Why some genes are retained in P&M?

- transmembrane proteins (complicated transport, folding – cotranslational incorporation to membrane)- high level and rapid protein synthesis

DNA transfer from organelles to nucleus is still active!

- plant nuclear genomes contain high amount of plastid DNA- even recent integrations (whole copies of plastid genomes)- in rice totally more than 800 kbp

Transfer of genes from organelles

high frequence of transgene transfer from plastids to nucleus

- in somatic cells 1 of 18.000- v pollen (plastid degradation!) 1 of 11.000- in egg cells 1 of 250.000

Heritability of organelle DNA

- usually uniparental (maternal), - various mechanisms:

– Chlamydomonas: paternal organelle DNA eliminated through methylation disabling its replication (otherwise methylation of DNA and RNAi do not occur)

– some higher plants – paternal plastids eliminated upon fertilization

Plastid DNA (cpDNA)

• dsDNA, circular (likely basic from)• lower G-C content (compared to nucleus)• high copy number (~30-100) per plastid• 20-40 organelles/nucleus• without histones, but Hu proteiny (Hu),

organized to nucleoids • 10-20 % of total leaf DNA

Typical cp genome – basic arrangement

Circle divided into „long“ and „short“ unique regions (LSC and SSC) separated with IR (recombination = inversion)

rRNA (rrn) and tRNA (trn) genes in clusters (like in E. coli)

Structural complexity of plastid DNA

Table 1. Frequency of Different cpDNA Structures across All Experiments in Three Species

No. of Observations

Structurea Arabidopsis Tobacco Pea

Circular 126 (42%) 524 (45%) 59 (25%) Linear 68 (23%) 250 (22%) 85 (36%) Bubble/D-loop 25 (8%) 67 (6%) 5 (2%) Lassolike 34 (11%) 115 (10%) 21 (9%) Unclassifiedb 44 (16%) 203 (17%) 66 (28%) a Each classification represents all molecules of that type regardless of size. b DNA fibers that were coiled or folded and could not be classified

[Lilly et al. Plant Cell. 13:245]

Sizes of plastid genome

• 70 - 200kb

• higher plants 120 – 170 kb

Sizes of mitochondrial genome

• S. cerevisiae 84 kb• mammels 16 kb• similar coding capacity

• higher plants – hunders kb (x weed – small chondriome 16 kb)

Economization in evolution x evolutionary trap in higher plants?

Mitochondrial genome

Mitochondrial genome

Maize: Zea maysseveral circle molecules

„master“ - 570 kb + subgenomic molecules derived from the „master“

Subgenomic circles through recombination between repeats (arrows)

Complexity of mt DNAreason?

Backert et al. Trends Plant Sci 2:478

Expression of chloroplast genome Genes mainly in operons – cotranscription

- processed to shorter segments (present as stable ribonucleoprotein units)

Higher plants plastids – approx. 30 transcription units (with promoter and terminater)- two polymerases PEP, NEP (plastid, nucleus-encoded pol.)

- promoters for PEP similar to bacterial (–10 and –35 sequence)- mostly promoters for both polymerases

transcript usually without cap and polyA, exceptionally editing

psbB

psbT psbH

petB

Intron

Intron

petD

psbN

Polycistronic RNA

Monocistronic RNA

Transcriptional units of plastid genome

Model of expression of psbB operon

Barkan A. Plantphysiol 2011;155:1520-1532

- Important role of pentatricopeptide-repeat proteins (also involved in editing)

Expression of mitochondrial genome

- transcripts without cap and polyA

- transkripts frequently edited

RNA Editing– discovered in plant mitochondrial genes– rare in plastids of higher plants

Definition: any process (except splicing) causing

change in RNA sequence (it is no more fully complementary to the template DNA sequence)

Editing of mitochondrial transcripts

• Many mitochondrial transcripts (tRNA, protein-coding)• Mainly C to U• Guide RNA and editosom (role of PPR, mechanism?)

Exchange of C to U

cytosin deaminase or replacement of nucleotide base

Introns in organelle genes

• orthologous genes can have different introns in the same positions

• same or similar introns in various genes and species (introns I. and II. type)

• obtained and lost repeatedly during evolution

Expression of plastid genes- PEP and NEP – (Plastid/Nuclear Encoded Polymerase)

- mutually regulated, - widely overlaping expression (double promoters) - not substitutible (both necessary)

- nuclear encoded sigma factors of PEP complex - some common for both plastids and mitochondria, - transcription usually induced by more than one factor

σ

Transcriptional regulation of plastid gene expression

1. Global – increase/decrease in expression of majority

of genes in the same time

2. Gene specific

sigma factors of PEP (procaryotic type)

e.g. psbD/psbC expression activated with light

Factors effecting chloroplast gene expression

Retrograde signalling- regulation of nuclear expression by signals from

P&M (products targeted to the organelles)

- response to changing conditions- full expression only if fully functional plastids are present

Signals: - chlorophyll biosynthesis precursors- electron transport components- redox signals + ROS- certain metabolites

Genes Encoded in the Chloroplast Genomes in Higher Plants

Gene Designation Gene Product I. Genetic System Chloroplast RNA genes

rDNA Ribosomal RNAs (16S, 23S, 4.5S, 5S)trn Transfer RNAs (30 species)

Gene transcriptionrpoA, B, C RNA polymerase , , ’ subunitsssb ssDNA-binding protein

Protein synthesisrps2,3,4,7,8,11 30S ribosomal proteins (CS) 2, 3, 4, 7, 8, 11rps12, 14, 15, 16, 18, 19 CS12, 14, 16, 18, 19rpl2, 14, 16, 20, 22 50S ribosomal proteins (CL) 2, 14, 16, 20, 22infA Initiation factor I

II. Photosynthesis Photosynthetic proteins

rbcL RUBISCO large subunitatpA, B, E ATP synthetase CF1, , subunitsatpF, H, I ATP synthetase CF0I, III, IV subunitspsaA, B, C Photosystem I A1, A2, 9-kDa proteinpsbA, B, C, D, E Photosystem II D1, 51 kDa, 44 kDa, D2, Cytb559-

9kDapsbF, G, H, I Photosystem II Cytb559-4kDa, G, 10Pi, I

proteinspetA, B, D Electron transport Cytf, Cytb6, IV subunits

Respiratory proteinsndhA, B, C, D NADH dehydrogenase (ND) subunits 1, 2, 3, 4ndhE, F NDL4L, 5

III. Others Maturase matK

Protease clpPEnvelope membrane protein cemA