Organelle genomesOrganelle gene expression processesOrganelle-to-nucleus signaling (retrograde regulation)

PCB6528 Plant Cell and Developmental Biology Spring 2012

Organelle genomes, gene expression and signaling

Christine Chase – 2215 Fifield Hall – 352-273-4862

cdchase@ufl.edu

Describe the organization and coding content of plant plastid and mitochondrial genomes

Discuss the similarities and differences between the plastid and plant mitochondrial genomes with respect to organization and evolution

Explain why plastid or mitochondrial genome coding content is not necessarily identical between plant species

Discuss the possible reasons that plant organelles retain genomes at all

Describe the process of plastid genome transformation

Discuss the utility and applications of plastid transformation and provide some specific examples

Objectives - Organelle genomes:

Small but essential genomes

Multiple organelles per cell; multiple genomes per organelle (20 – 20,000 genomes per cell, depending on cell type)

Organized in nucleo-protein complexes called nucleoids

Non-Mendelian inheritance; usually but not always maternally inherited in plants

Encode necessary but insufficient information to elaborate a fully functional organelle

Many nuclear gene products required for organelle function

translated on cytosolic ribosomes &imported into the organelles

plant mitochondria also import tRNAs needed for a complete set!

Considerable cross-talk between nuclear and organelle genetic systems

Organelle genomes

Comparative sizes of plant genomes

Genome Size in bpArabidopsis thaliananuclear

1.4 x 10 8

Arabidopsis thalianamitochondria

3.7 x 10 5

Arabidopsis thalianaplastid

1.5 x 10 5

Zea maysnuclear

2.4 x 10 9

Zea maysmitochondria

5.7 x 10 5

Zea maysplastid

1.4 x 10 5

Target P prediction analysis of the complete Arabidopsis nuclear genome sequence (Emanuelsson et al., J Mol Biol 300:1005)says .....

~ 10% of the Arabidopsis nuclear genome (~2,500 genes) encode proteins targeted to the mitochondria

~ 14% of the Arabidopsis nuclear genome (~3,500 genes) encodes proteins targeted to the plastid

So 25% of the Arabidopsis nuclear genome is dedicated to organelle function!

Proteome reflects metabolic diversity of these organelles, both anabolic and catabolic

Organelle genomics & proteomics

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Endosymbiont origin of organelles Original basis in cytologyConfirmation by molecular biology α proteobacteria as closest living relatives to mitochondriaCyanobacteria closest living relatives to plastidsArchaebacteria considered to be related to primitive donor of the nuclear genome

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[Gillham 1994 Organelle Genes and Genomes]

Chimeric origin of eukaryotic nuclear genomes

Genes per category among

383 eubacterial- & 111

archeaebacterial- related genes in the

yeast nuclear genomeEsser et al. 2004

Mol Biol & Evol 21:1643

Evolution of the eukaryotic genomes

Reduced coding content of organelle genomes

• Functional gene transfer to nucleus with protein targeted back to organelle

• Functional re-shuffling - organelles replace prokaryotic features with eukaryotic, “hybrid” or novel features

Evolution of mitochondrial genome coding content Genome Protein

coding genes

Rikettsia prowazekii (smallest proteobacterial genome)

832

Reclinomonas americana mitochondria(protozoan; most mitochondrial genes)

62

Marchantia polymorpha mitochondria1.9 x 10 5 bp(liverwort, non-vascular plant )

64

Arabidopsis thaliana mitochondria3.7 x 10 5 bp(vascular plant)

57

Homo sapiens mitochondria 13

Evolution of plastid genome coding content Genome Protein

coding genes

Synechococcus (cyanobacteria) 3,300Paulinella chromatophoraphotosynthetic body(endosymbiont cyanobacteria)

867

Porphyra purpurea plastid(red alga)

209

Chlamydomonas reinhardtii plastid(green alga)

63

Marchantia polymorpha plastid(liverwort, non-vascular plant)

67

Arabidopsis thaliana plastid(vascular plant)

71

Epifagus virginiana plastid (non-photosynthetic parasitic plant)

42

Functional gene transfer from organelle to nuclear genome

• Gene by gene • Likely occurs via RNA intermediates • Evidence for frequent and recent

transfers in plant lineage • Results in coding content differences

among plant organelle genomes

• What is required for a functional gene re-location from organelle to nucleus?

• How would we know this occurs via RNA intermediates?

• Southern blot hybridization of total cellular DNA samples

• Mitochondrial nad1 and rps10 probes • Shading = taxa with no hybridization to rps10 • Bullets = taxa with confirmed nuclear copy of

rps10

[Adams et al. Nature 408:354]

• Why is there no hybridization of rps10 probes to DNA samples with confirmed nuclear copy of rps10?

(Hint: How are the relative genome copy numbers and sizes exploited in this screen?) • What is the purpose of the nad1 probe?• What are the consequences of these events

with respect to plant mitochondrial genome coding content?

Functional gene transfer: Recent repeated transfers of the plant mitochondrial rps10 to the nucleus

Reduced plastid genome content in nonphotosynthetic plastids:

Parasitic plants with 70-20 kb plastomes have lost photosynthetic genes, some ribosomal proteins & some tRNAs

Essential tRNA hypothesis: [Barbrook et al. Trends in Plant Sci. 11:101]Plastid tRNAs needed to support mitochondrial function• tRNA-Glu as precursor for the synthesis of

heme for mitochondrial respiratory electron transfer

• tRNA-Met imported into mitochondria for mitochondrial protein synthesis

Epifagus virginiana (beechdrops)A non-phoptosynthetic, parasitic plant Has a plastid genome of 71 kb encoding 7 tRNAs and 2 ribosomal proteins

http://2bnthewild.com/plants/H376.htm

Land Plant Plastid Genome Organization

Physical map (e. g. restriction map or DNA sequence) indicates a 120-160 kb circular genome

Large inverted repeat (LIR) commonly 20-30 kb

• Large single copy (LSC) region • Small single copy (SSC) region

Active recombination within the LIR

Expansion and contraction of LIR• Primary length polymorphism among land

plant species• 10-76 kb

Some conifers and legumes have very reduced or no LIR

Inversion polymorphisms within single copy regions mediated by small dispersed repeats

(Maier et al. J Mol Biol 251:614)

Plastid genome organization

Plastid genes in operons

(from Palmer [1991] in Cell Culture and Somatic Cell Genetics of Plants, V 7A. L Bogorad and IK Vasil eds. Academic Press,

NY, pp 5-142)

Recombination across inverted repeatsleads to inversions

How can these inversion isomers be detected?

trn N

rps19

rps15

psbA

ndhF

ndhBtrn N

ndhBrps19

rpl22

trn N

rps19

rps15

psbA

ndhF

ndhBtrn N

ndhBrps19

rpl22

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

Fiber FISH of tobacco plastid DNA

Structural Plasticity of cpDNA Molecules from Tobacco, Arabidopsis, and Pea

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

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]

Structural Plasticity of cpDNA Molecules from Tobacco, Arabidopsis, and Pea

Plastid genome coding content

Chloroplast Genome Database:http://chloroplast.cbio.psu.edu/ (Cui et al., Nucl Acids Res 34: D692-696)

Generally conserved among land plants, more variable among algae

Genes for plastid gene expression rRNAs, tRNAs ribosomal proteins RNA polymerase

Genes involved in photosynthesis 28 thylakoid proteins

Photosystem I (psa)Photosystem II (psb)ATP synthase subunits (atp)NADH dehydrogenase subunits (nad)Cytochrome b6f subunits (pet)

RUBISCO large subunit (rbcL)(rbcS is nuclear encoded)

Plastid genomes encode integral membrane components of the

photosynthetic complexes

Photosynthetic composition of the thylakoid membraneGreen = plastid-encoded subunitsRed = nuclear-encoded subunits

• What do you notice about the plastid vs nuclear-encoded subunits ?

• What hypotheses does this suggest regarding the reasons for a plastid genome?

[Leister, Trends Genet 19:47]

Plastid genome transformation

DNA delivery by particle bombardment or PEG precipitation

DNA incorporation by homologous recombination

Initial transformants are heteroplasmic, having a mixture of transformed and non-transformed plastids

Selection for resistance to spectinomycin (spec) and streptomycin (strep) antibiotics that inhibit plastid protein synthesis

Spec or strep resistance conferred by individual 16S rRNA mutations

Spec and strep resistance conferred by aadA gene (aminoglycoside adenylyl transferase)

Untransformed callus bleached; transformed callus greens and can be regenerated

Multiple selection cycles may be required to obtain homoplasmy (all plastid genomes of the same type)

Plastid genome transformation

[Bock & Khan, Trends Biotechnol 22:311]

Selection for plastid transformants

[Bock , J Mol Biol 312:425]

A) leaf segments post bombardment with the aadA geneB) leaf segments after selection on spectinomycin; C) transfer of transformants to spectinomycin + streptomycin D) recovery of homoplasmic spec + strep resistant transformants

Applications of plastid genome transformation by homologous recombination

[Bock, Curr Opin Biotechnol 18:100]

[Hager et al. EMBO J 18:5834]

Functional analysis of plastid ycf6 in transgenic plastids

Functional analysis of plastid ycf6 in transgenic plastids

[Hager et al. EMBO J 18:5834]

ycf6 knock-out lines:•Homoplasmic for aadA insertion into ycf6•Pale-yellow phenotype•Normal PSI function and subunit accumulation

•Normal PSII function and subunit accumulation

•Abnormal b6f (PET) subunit accumulation •Mass spectrometry demonstrates YCF6 in normal plastid PET complex

Why, if ycf6 is the disrupted gene,does another PET complex subunit (PETA) fail to accumulate ?

Non-Functional DNA transfer from organelle to nuclear genome

Frequent

Continual (can detect in “real-time” as well as evolutionary time)

In large pieces

e.g. Arabidopsis 262 kb numtDNA (nuclear-localized mitochondrial DNA)

88,000 years ago

e.g. Rice 131 kb nupDNA (nuclear-localized plastid DNA)

148,000 years ago

Non-functional plastid-to-nucleus DNA transfer

• Transform plastids with:plastid promoter – aadA

linked to nuclear promoter - neo

• Pollinate wild-type plants with transformants

• % seed germination on kanamycin ~ frequency of nuclear promoter - neo

transferred from plastid to nucleus

Why does this experiment primarily estimate the frequency of DNA transfer from plastid to nucleus, rather than the frequency of functional gene transfer from plastid to nucleus?

How would you re-design the experiment to test for features of a functional gene transfer?

[Timmis et al. Nat Rev Genet 5:123]

Land Plant Mitochondrial Genome Organization

208-2400 kb depending on species

Relatively constant coding but highly variable organization among and even within a species

Physical mapping with overlapping cosmid clones

• Entire complexity maps as a single “master circle”

• All angiosperms except Brassica hirta have one or more recombination repeats.

• Repeats not conserved among species

• Direct and/or inverted orientations on the “master”

• Recombination generated inversions (inverted repeats)

• Recombination generated subgenomic molecules (deletions) (direct repeats), some present at very low copy number (sublimons)

• Leads to complex multipartite structures

Recombination across direct repeats leads to deletions (subgenomic molecules)

a’b’

c

d

Pac I

PmeI

a b c d

b’ c’ d’a’

Pac I AscI

abc’

d’

Not I

AscIHow can these deletion (subgenomic) isomers be detected?

a’b’c’d’

a b c d

Pac I

AscI

PmeINot I

b’c’d’ a’

Arabidopsis mitochondrial genome organization> >>>

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[modified from Backert et al. Trends Plant Sci 2:478]

Two pairs of repeats active in recombination• One direct (magenta, top left)• One inverted (blue, top left)

Recombining the inverted (blue pair) creates an inversion

• What has happened to the orientation of the magenta repeats (top right)?

(Backert and Börner, Curr Genet 37:304)

Branched rosette and linear molecules from C. album mitochondria

[Backert et al. Trends Plant Sci 2:478]

Structural plasticity of plant mitochondrial DNA

Structural plasticity of plant organelle genomes

Plastid genomes map as a single circle • Inversion isomers• Indicate recombination through the LIR

Plant mitochondrial genomes map as a single master circle plus

• Many subgenomic circles • Inversion isomers• Imply recombination through multiple

direct& inverted repeat pairs

Direct visualization via EM or FISH • Rosette/knotted/branched structures• Longer-than genome linear molecules• Shorter-than genome linear and circular

molecules• Sigma molecules• Branched linear molecules• Few if any genome-length circular

molecules (mitochondria only)

Circular maps – linear molecules

fixed terminal redundancy (e.g. phage T7)ABCDEF______________XYZABC

circularly permuted monomers ABCDEF______________XYZ BCDEF______________XYZA CDEF _____________ XYZAB

circularly permuted monomers & terminal redundancy (e.g. phage T4) CDEF______________XYZABCDEF DEFG____________ XYZABCDEFG EFGH___________XYZABCDEFGH

linear dimers or higher multimersABCDEF__________XYZABCDEF_________XYZ

A Z B

Y C

X D

In a circular molecule or map, fragment A is linked to B, B to C, C to D, D to X, X to Y, Y to Z and Z to A. But these linkages also hold true for linear molecules

[Freifelder, 1983, Molecular Biology]

Physical structures of DNA obtained via rolling circle DNA replication

Recombination initiated DNA replication

[Kreuzer et al. J Bacteriol 177:6844]

Complex rosette/knotted structures• nucleoids

Longer-than genome linear molecules• rolling circle replication• intermolecular recombination of linear

molecules

Shorter-than genome linear and circular molecules

• intramolecular recombination between direct repeats

Sigma molecules• rolling circles• recombination of circular & linear molecules

Branched linear molecules• recombination• recombination-mediated replication

Few if any genome-length circular molecules • limited number of circular rolling circle

replication templates

Possible origins of structural plasticity in plant organelle genomes

Plant mitochondrial genome coding content

In organello protein synthesis estimates 30-50 proteins encoded by plant mitochondrial genomes

Complete sequence of A. thaliana mit genome 57 genes respiratory complex componentsrRNAs, tRNAs, ribosomal proteinscytochrome c biogenesis

Plant mit genomes lack a complete set of tRNAs

mit encoded tRNAs of mit originmit encoded tRNAs functional transfer from

the plastid genomenuclear encoded tRNAs imported into

mitochondria to complete the set

42 orfs that might be genes

Gene density (1 gene per 8 kb) lower than the nuclear gene density (1 gene

per 4-5 kb)!

Plant mitochondrial genome coding content

Table 3 General features of mtDNA of angiosperms

Feature Ntaa Ath Bna Bvu OsaMC (bp) 430,597 366,924 221,853 368,799 490,520A+T content (%) 55.0 55.2 54.8 56.1 56.2Long repeated (bp) b 34,532 11,372 2,427 32,489

127,600Uniquec 39,206 37,549 38,065 34,499 40,065Codingd (9.9%) (10.6%) (17.3%) (10.3%) (11.1%)Cis-splicing introns 25,617 28,312 28,332 18,727

26,238 (6.5%) (8.0%) (12.9%) (5.6%) (7.2%)

ORFse 46,773 37,071 20,085 54,288 12,009 (11.8%) (10.4%) (9.2%) (16.1%) (3.3%)

cp-derived (bp) 9,942 3,958 7,950 g 22,593 (2.5%) (1.1%) (3.6%) 2.1% h (6.2%)

Others 274,527 248,662 124,994 262,015 (69.3%) (69.9%) (57%) 65.9% (72.2%)

Gene contentf 60 55 53 52 56

(from Sugiyama et al. Mol Gen Gen 272:603)

Mitochondrial genomes encode integral membrane components of

the respiratory complexes

= one mitochondria-encoded subunit *

IIAOX

intermembrane space

innermembrane

matrix

I

UQH2

UQ

H+

CYC

IV

H+

III

H+

H+

ATPSynthase

II

TCA cycle NADH

NAD+

NAD(P)H DH external

NAD(P)H DH internal

2H2O

O2

2H2O

O2 ADP ATP

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There is some species-to-species variation with respect to the presence or absence of genes encoding respiratory chain subunits. What is the likely explanation for this observation?

(Modified from Rasmusson et al. Annu Rev Plant Biol 55:23)