PCB6528 Plant Cell and Developmental Biology Spring 2016 signaling

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PCB6528 Plant Cell and Developmental

Biology Spring 2016

Organelle genomes, gene expression and signaling http://www.hos.ufl.edu/sites/default/files/courses/ pcb6528/PCB6528S16_outline.htm

Christine Chase – 2215 Fifield Hall –

352-273-4862 cdchase@ufl.edu

How nuclear and cytoplasmic genetic systems co-evolved and now interact to build and maintain highly abundant and complex respiratory and photosynthetic factories

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

Objectives - Organelle genomes:

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

Reconcile discrepancies between physical maps and the observed DNA structures

Describe how evolution has shaped and changed modern-day organelle genome coding content compared to ancestral prokaryotic genomes

Consider the possible reasons that plant organelles retain genomes at all!

Explain the challenges and experimental opportunities associated with genetic transformation of the plastid genome

Organelle genomes

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

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

Comparative sizes of plant genomes

Genome

Arabidopsis thaliana nuclear

Arabidopsis thaliana mitochondria

Arabidopsis thaliana plastid

Zea mays nuclear

Zea mays mitochondria

Zea mays plastid

Size in bp

1.4 x 10 8

3.7 x 10 5

1.5 x 10 5

2.4 x 10 9

5.7 x 10 5

1.4 x 10 5

Organelle genomics & proteomics

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

Endosymbiont origin of organelles

*

*

*

Original basis in cytology

Confirmation by molecular biology

α proteobacteria as closest living relatives to mitochondria

Cyanobacteria closest living relatives to plastids

Archaebacteria considered to be related to primitive donor of the nuclear genome

*

*

*

[Gillham 1994

Organelle Genes & Genomes]

Eukaryotic nuclear genomes – a big mix & match experiment

383 eubacterial - &

111 archeaebacterial related genes in the yeast nuclear genome

Genes per category

Esser et al. 2004 Mol

Biol & Evol 21:1643

Evolution of mitochondrial genome coding content

Genome Protein coding genes

832 Rikettsia prowazekii

(smallest  proteobacterial genome)

Reclinomonas americana mitochondria

(protozoan)

Marchantia polymorpha mitochondria

1.9 x 10 5 bp

(liverwort, non-vascular plant )

Arabidopsis thaliana mitochondria

3.7 x 10 5 bp

(vascular plant)

Saccharomyces cerevisiae

7.8 x 10 4 bp

(yeast)

Homo sapiens mitochondria

62

64

57

11

13

Evolution of plastid genome coding content

Genome

Synechococcus (cyanobacteria)

Paulinella chromatophora photosynthetic body

(endosymbiont cyanobacteria)

Porphyra purpurea plastid

(red alga)

Chlamydomonas reinhardtii plastid

(green alga)

Marchantia polymorpha plastid

(liverwort, non-vascular plant)

Arabidopsis thaliana plastid

(vascular plant)

Epifagus virginiana plastid

(non-photosynthetic parasitic plant)

Protein coding genes

3,300

867

209

63

67

71

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 modern-day plant organelle genomes!

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

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

The structures of nuclear genes encoding mitochondrial RPS10

• Triangles = nuclear intron positions

• Shading = mitochondrial targeting sequences

• Stripes = regions homologous to other known nuclear genes.

• Arrow = continuing open reading frame

• What do these diverse gene structures, along with phylogeny, tell us about the functional transfer of the rps10 gene from mitochondria to nucleus?

29 of the 31 mitochondria-located rps10 genes examined in this study contain a conserved intron, which is missing in all of the nuclear-located rps10 genes. What does this suggest about the mechanism of functional gene transfer from mitochondria to nucleus?

[Adams et al. Nature 408:354]

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

Plastid genome organization

(Maier et al. J Mol Biol

251:614)

Plastid ATP synthase genes in operons

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

(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)

The plastid genome oversimplified : recombination across inverted repeats leads to inversions

rps15

trn N

ndhB

rps19

psbA

ndhF

trn N

ndhB

rps19

psbA

ndhF

trn N

ndhB

rps19

rpl22 How can these inversion isomers be distinguished?

rps15

trn N

ndhB

rps19

rpl22

Fiber FISH of tobacco plastid DNA

IR probe SSC+IR probe

SC gene probes

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

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

IR probe

IR probe

SSC+IR probe

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

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

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

Species

No. of Observations

Structure a Arabidopsis Tobacco Pea

Circular

Linear

126 (42%)

68 (23%)

524 (45%)

250 (22%)

59 (25%)

85 (36%)

Bubble/D-loop

Lassolike

25 (8%)

34 (11%)

67 (6%)

115 (10%)

5 (2%)

21 (9%)

Unclassified b 44 (16%) 203 (17%) 66 (28%) b a Each classification represents all molecules of that type regardless of size.

DNA fibers that were coiled or folded and could not be classified

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

Land plant mitochondrial genome organization

208-2400 kb depending on species

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

• Almost no conservation of gene order among 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 maps (and structures)

Recombination across direct repeats leads to deletions (subgenomic molecules) a b c

Not I d

PmeI

AscI

Pac I a b c d

Pac I b’ c’ d’

AscI

Not I

Pac I b’ a’ a b d’ c’ d c

AscI

PmeI

How can these deletion (subgenomic) isomers be distinguished?

Arabidopsis mitochondrial genome organization 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) While physical mapping yields the organizations shown in a, optical mapping shows a very different organization

[Gualberto et al. Biochimie 100:107]

Branched rosette and linear molecules from

C. album mitochondria

(Backert and Börner, Curr Genet 37:304)

Structural complexity of plant mitochondrial

DNA

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

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

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 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 multimers

ABCDEF__________XYZABCDEF_________XYZ

Physical structures of DNA obtained via rolling circle DNA replication

[Freifelder, 1983, Molecular Biology]

Circular maps from linear molecules

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 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 multimers

ABCDEF__________XYZABCDEF_________XYZ

Recombination dependent DNA replication

[RDR]

[Marechal and Brisson New Phytol 186:299]

Origins of plant organelle genome complexity

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 -mediated replication

Few genome-length circular molecules

(none for mitochondrial)

• What governs the stable inheritance of this mess?

Recombination and plant organelle genome stability

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 wild-type recombination partners far out-number a new mutant !

Genome replication

• structures support the recombination dependent replication model

? Does recombination also create a cohesive unit of inheritance

Recombination and plant organelle genome

(in) stability

Recombination surveillance

• Prevents or limits recombination between short repeats (~100-500 bp) in plant organelle DNAs

Mediated by four nuclear-encoded protein families

• Dual-targeted or duplicate genes encode pt- & mt-targeted versions

• MSH1 - Eubacterial mismatch repair homologs

• RECA - Eubacterial recombination homology search & strand invasion

• OSB - Recently evolved plant-specific organelle single-stranded DNA binding proteins

• Whirly - Recently evolved, primarily plant, single-stranded DNA binding proteins

Plant organelle recombination surveillance

DSB in mtDNA

RECA, ODB/RAD52

WHY

RECA, MSH1,

OSB homologous recombination/ gene conversion

RECA-independent recombination at micro-homologies

Mutations in any surveillance gene destabilize mitochondrial and/or plastid genome organization

[Modified from Gualberto et al. Biochimie 110:107]

Down-regulation of dual-targeted MSH1 alters organelle function and genome organization

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

• Organelle recombination is regulated

• Regulated homologous recombination important for genome replication and repair

• De-regulation destabilizes organelle genome organization with phenotypic consequences

[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 membrane

Green = plastid-encoded subunits

Red = 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 components rRNAs, tRNAs, ribosomal proteins cytochrome c biogenesis

Plant mit genomes lack a complete set of tRNAs mit encoded tRNAs of mit origin mit encoded tRNAs functional transfer from the plastid genome nuclear 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 Nta a 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 32,489 127,600

Unique c

Coding d

39,206 37,549 38,065 34,499 40,065

(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%)

ORFs e cp-derived (bp)

Others

Gene content f

46,773 37,071 20,085 54,288 12,009

(11.8%) (10.4%) (9.2%) (16.1%) (3.3%)

9,942 3,958

(2.5%) (1.1%)

7,950 g 22,593

(3.6%) 2.1% h (6.2%)

274,527 248,662 124,994 262,015

(69.3%) (69.9%) (57%) 65.9% (72.2%)

60 55 53 52 56

Gene content is similar but NOT identical. Why?

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

Mitochondrial genomes encode integral membrane components of the respiratory complexes

NAD(P)H DH external

H

+ inner ***

I

NAD(P)H DH internal

H

+

CYC

H

+ intermembrane space

UQH

2

UQ

*

TCA

NAD+ cycle NADH

AOX

*

III IV

***

O

2

2H

2

O

H

+

2H

2

O

O

2

Synthase

ADP ATP

* matrix

* = one mitochondria-encoded subunit

(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

A) leaf segments post bombardment with the

aadA gene

B) leaf segments after selection on spectinomycin

C) transfer of transformants to spectinomycin + streptomycin

D) recovery of homoplasmic spec + strep resistant transformants

[Bock , J Mol Biol 312:425]

Applications of plastid genome transformation by homologous recombination

[Bock , Curr Opin Biotechnol 18:100]

Applications of plastid genome transformation by homologous recombination

[Bock , Curr Opin Biotechnol 18:100]

Applications of plastid genome transformation by homologous recombination

[Bock , Curr Opin Biotechnol 18:100]

Functional analysis of plastid ycf6 in transgenic plastids

[Hager et al.

EMBO J

18:5834]

Functional analysis of plastid ycf6 in transgenic plastids 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 ?

[Hager et al. EMBO J 18:5834]

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]

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