Organelle genomes Organelle gene

expression Organelle

signaling

PCB6528 Plant Cell and Developmental Biology Spring 2015

Organelle genomes, gene expression and signaling

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

cdchase@ufl.edu

How do we build and maintain these highly abundant and complex respiratory and photosynthetic factories?

[Leister, Trends Genet 19:47; Salvato, Plant Physiol 164:637]

Describe the physical organization and coding content of modern-day organelle genomes

Explain how we can reconcile discrepancies between physical maps and the observed DNA structures

Describe how evolution has shaped and changed modern-day organelle genomes from their ancestral prokaryotic genomes

Discuss the possible reasons that plant organelles retain genomes at all

Discuss the challenges associated with genetic transformation of organelle genomes and how these challenges have been met to achieve genetic transformation of the plastid genome

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

Objectives - Organelle genomes:

Organelle genome databases:http://www.hsls.pitt.edu/obrc/index.php?page=organelle

Small but essential

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 maternal

Necessary but not sufficient to elaborate a functional organelle• nuclear gene products required• translated on cytosolic ribosomes• imported into the organelles• plant mitochondria also import

tRNAs

Organelle genomes

Comparative sizes of plant genomes

Genome Size in bp

Arabidopsis 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

*

*

*[Gillham 1994

Organelle Genes & Genomes]

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

***

Eukaryotic nuclear genomes – a big mix & match experiment

Esser et al. 2004 Mol Biol & Evol

21:1643

383 eubacterial- & 111

archeaebacterial- related genes in the

yeast nuclear genome

Genes per category

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) 3,300

Paulinella 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

Evolution of the eukaryotic genomes

Reduced coding content of organelle genomes compared to endosymbiont

Modern day organelle genomes don’t encode all the proteins required for organelle function

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

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

Functional gene transfer from organelle to nuclear genome

• Gene by gene • 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?

• Southern blot hybridization of total cellular DNA

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

gene• Why no hybridization of rps10 probes to DNA

with confirmed nuclear copy? (Hint: How are the relative genome copy numbers exploited in this screen?)

• What is the purpose of the nad1 probe?• What are the implications of these findings for

plant mitochondrial genome coding content?

[Adams et al. Nature 408:354]

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

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

Land Plant Plastid Genome Organization

120-160 kb depending on species• conserved coding • conserved physical organization

Physical map • restriction map or DNA sequence • 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

SC region inversion polymorphisms mediated by infrequent recombination between small dispersed repeats

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

Plastid genome organization

Plastid ATP synthase 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)

Note we do not see this for plant mitochondrial genomes, where gene order is highly scrambled!

The plastid genome oversimplified:recombination across inverted repeats

leads to inversions

How can these inversion isomers be distinguished?

trn N

rps19

rps15

psbA

ndhF

ndhB

trn N

ndhB

rps19

rpl22

trn N

rps19

rps15

psbA

ndhF

ndhB

trn N

ndhB

rps19

rpl22

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

Fiber FISH of tobacco plastid DNA

IR probe SSC+IR probe

SC gene probes

Structural complexity of plastid DNA from tobacco, arabidopsis, and pea

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

IR probe

IR probe

SSC+IR probe

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 complexity of plastid DNA from tobacco, arabidopsis, and pea

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

ab c d

b’ c’ d’a’

Pac IAscI

ab

c’

d’

Not I

AscI

How can these deletion (subgenomic) isomers be distinguished?

a’b’c’d’

ab c d

Pac I

AscI

PmeI

Not I

b’c’d’ a’

Arabidopsis mitochondrial genome organization

[Gualberto et al. Biochimie 100:107]

a) Two pairs of repeats active in recombination• One direct (A, top left)• One inverted (B, top left)

Recombining the inverted pair creates an inversion• What happens to the B pair ?

Recombining the direct pair creates a deletion (2 subgenomes) that can further recombine

b) Although physical mapping yields the organizations shown in a, optical mapping shows a very different organization

(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 complexity of plant mitochondrial DNA

Structural complexity 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 for mitochondria

Circular maps from 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

Circular maps from 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

Recombination dependent DNA replication[RDR]

[Marechal and Brisson New Phytol 186:299]

Complex rosette/knotted structures• nucleoids

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

linear moleculesShorter-than genome linear and circular molecules

• intramolecular recombination between direct repeats

Sigma molecules• rolling circles• recombination of circular & linear

moleculesBranched linear molecules

• recombination-mediated replication

Few genome-length circular molecules (none for mitochondrial)

• What governs the stable inheritance of this mess?

Origins of plant organelle genome complexity

Repair of DNA damage• organelles rich in damaging ROS• low rates of synonymous-substitution

• homologous recombination with gene conversion

repair point mutations repair DNA breaks

• lots of wild-type recombination partners

Genome replication• structures support the recombination dependent replication model

? Does recombination also create a cohesive unit of inheritance

Recombination and plant organelle genome stability

Recombination surveillance• Limits recombination between short repeats (~100-500 bp) in plant organelle DNAs

Mediated by four protein families• members targeted to plastids &/or mitochondria

• MSH1 - Eubacterial mismatch repair homologs

• RECA - Eubacterial recombination homology search/strand invasion

• OSB - Plant-specific organelle single-stranded DNA binding proteins

• Whirly - Primarily plant, single-stranded DNA binding proteins

Recombination and plant organelle genome (in) stability

Plant organelle recombination surveillance team

[Marechal and Brisson New Phytol 186:299]

Down-regulation of MSH1 alters organelle function and genome

organization

Mitochondrial genome reorganization left, co-segregating with leaf variegation, right

Organelle recombination is regulated

De-regulation destabilizes organelle genome organization with phenotypic consequences

Some recombination is good; too much is bad!

[Sandhu et al. Proc Natl Acad Sci USA 104:1766]

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]

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 Osa

MC (bp) 430,597 366,924 221,853 368,799 490,520

A+T content (%) 55.0 55.2 54.8 56.1 56.2

Long repeated (bp) b 34,532 11,372 2,427 32,489127,600

Uniquec 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)

Gene content is similar but NOT identical. Why?

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

******** *******

**

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

Plastid genome transformation

What are the special challenges for the genetic transformation of organelle genomes?

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 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]