plant energy biology

Plant Energy Biology

New knowledge research…

What is an ARC Centre of Excellence?

•Large Funded Centres From Federal Government

•Aimed to Achieve Scale and Focus

•Funded for Period of 5 years

•Only 19 in total in Australia - in all Areas of Scientific Research

What is Plant Energy Biology?

Only ARC Centre of Excellence in Western Australia

Four Chief Investigators

Jim Whelan

Harvey Millar

Steve Smith

Ian Small - will come from France to joint centre

What is Plant Energy Biology?

Funding $2.5 million a year for 5 years

Additional Funding From University and State Government = ~$10 Million

Total Funding = $22.5 Million

What is Plant Energy Biology?

Aim of Centre is to elucidate the mechanism(s) of control of energy

metabolism in cells by understanding the control switches and regulatory circuits

that control metabolism

Investigate master control switches controlling gene expression

for energy metabolism in cells

Achieve this using Functional Genomics, Genomics, Transcriptomics,

Proteomics, Metabolomics, Bio-informatics

Integrate all these approaches

Education Program

Honours

Scholarships = $6,000

Access to high level training in a variety of Disciplines

Ph. D. Program

Top up Scholarships ~ $18,000 + $7,000 = $25,000 (Tax free)

Annual training courses in various techniques and methods

Mitochondrial molecular biology 2

• evolution of mitochondria

• maternal inheritance of mtDNA

• mtDNA and human evolution

Summary of lecture 1

• mitochondria are essential for ATP synthesis in eukaryote cells

• mitochondria have their own DNA: small circular chromosomes

• human mtDNA has no non-coding regions and a unique organisation

• they replicate by fission, separately from the rest of the cell

• mtDNA encodes a few structural proteins, ribosomal proteins and tRNAs

• most mitochondrial proteins are encoded on nuclear genes

•animal and fungal mitochondria have a different genetic code (ie, non-universal)

Human mtDNA

• small, double stranded circular chromosome

• 16,569 bp in total

• no non-coding DNA

• no introns

• polycistronic replication which is initiated from the D (displacement)- loop region

• followed by splicing of transcript to form messages.

Organisation of the mitochondrial chromosome

human mtDNA

yeast mtDNA

Yeastmitochondrial chromosome

• 68-75 kb, similar in structure to bacterial genome

• contains introns and non-regions between genes.

• Same proteins made as in animals

• genes transcribed separately

Mitochondria replicate much like bacterial cells. When they get too large, they undergo fission. This involves a furrowing of the inner and then the outer membrane as if someone was pinching the mitochondrion. Then the two daughter mitochondria split. Of course, the mitochondria must first replicate their DNA. An electron micrograph depicting the furrowing process is shown in these figures.

Mitochondrial replication

cell division: random distribution of mitos between daughter cells

mitochondrialreplication

Evolution of mitochondria

Mitochondria are generally thought to have evolved endosymbiotically when an anaerobic prokaryotic cell engulfed an aerobic bacterium and formed a stable symbiosis. Loss of most of the aerobe’s genome to the nucleus of the host allowed the latter to control the former.

Endosymbiotic hypothesis of mitochondrial evolution

This hypothesis suggests that the animal mt genome is most highly evolved as it has lost more function than its yeast and plant counterparts. MtDNA from some protozoa show the closest homology to the “ancestral” mitochondrial genome.

Chloroplasts are thought to have arisen from cyanobacteria in a similar fashion.

Evolution of mitochondria

Evolution of mitochondria

Mitochondria are generally thought to have evolved endosymbiotically when an anaerobic eukaryote cell engulfed an aerobic bacterium and formed a stable symbiosis. Loss of most of the aerobe’s genome to the nucleus of the host allowed the latter to control the former.

endocytosishost membrane

Chloroplasts of plants and algae are thought to have arisen from endosymbiosis of a cyanobacterium

(blue-green alga)

Clues to the endosymbiotic origin of organelles come from studies of “modern” symbiotic relationships

- these can be either mutualistic or parasitic

- in symbioses where the microsymbiont lives inside the host cell, the asociation is referred to as endocytobiotic

- these associations have common structures around the endosymbiont.

The evidence for mitochondria and chloroplasts

• Both mitochondria and chloroplasts have their own protein-synthesizing machinery, and it resembles that of prokaryotes not that found in the cytoplasm of eukaryotes.

• Their ribosomal RNA (rRNA) and the structure of their ribosomes resemble those of prokaryotes, not eukaryotes.

The evidence for mitochondria and chloroplasts

• A number of antibiotics (e.g., streptomycin) that act by blocking protein synthesis in bacteria also block protein synthesis within mitochondria and chloroplasts. They do not interfere with protein synthesis in the cytoplasm of the eukaryotes.

• Conversely, inhibitors (e.g., diphtheria toxin) of protein synthesis by eukaryotic ribosomes do not have any effect on bacterial protein synthesis nor on protein synthesis within mitochondria and chloroplasts.

• The antibiotic rifampicin, which inhibits the RNA polymerase of bacteria, also inhibits the RNA polymerase within mitochondria. It has no such effect on the RNA polymerase within the eukaryotic nucleus.

• Mitochondria and chloroplast electron transport components show great sequence homology with bacterial and cyanobacterial components - these are not found elsewhere in the eukaryote cell.

Factors against the theory:

• Mitochondria and chloroplasts only code for a few proteins. Most of the proteins found in the organelles are actually coded for by the nuclear DNA. (Did the organelle DNA jump to the nuclear DNA in evolutionary history?)

• Mitochondrial and chloroplast DNA have introns, a phenomenon never seen in prokaryotes.(Did this characteristic jump from the nuclear DNA to the organelle DNA?)

• If the theory of endosymbiosis is true, then one must ask what was the original eukaryotic cell (without mitochondria or chloroplasts) and how did it survive (glycolysis?). Why have not any primitive eukaryotic cells ever be found that are devoid of these organelles (is today's eukaryote just too superior?)

• In modern symbioses, there is no good evidence for gene transfer between endosymbiont and the host.

Most mitochondrial proteins are encoded in the nucleus, synthesised in the cytosol and transported to the mitochondrion.

The highlighted labels are drugs that can be used to block the process and test the source of the mitochondrial protein.

Mitochondrial ribosomes have a similar structure to those of bacteria - ie, 70S (cf the cytosol which are 80S).

This enables mitochondrial protein synthesis to be distinguished from that in the cytosol using inhibitors such as chloramphenicol and cycloheximide.

Despite having their own genome, most mitochondrial proteins are encoded in the nucleus, made in the cytosol and imported into the mitochondria

In all organisms, only a few of the proteins of the mitochondrion are encoded by mtDNA, but the precise number varies between organisms

• Subunits 1, 2, and 3 of cytochrome oxidase• Subunits 6, 8, 9 of the Fo ATPase• Apocytochrome b subunit of complexIII• Seven NADH-CoQ reductase subunits (except in yeast)

The nucleus encodes the remaining proteins which are made in the cytosol and imported into the mitochondrion.

Most of the lipid is imported.

Synthesis of mitochondrial proteins

Mitochondria are largely maternally inherited in higher Mitochondria are largely maternally inherited in higher animals and plantsanimals and plants

In mammals, most of the mitochondrial DNA (mtDNA) is inherited from the mother.  This is because the sperm carries most of its mitochondria its tail and has only about 100 mitochondria compared to 100,000 in the oocyte.  

Although sperm mitochondria penetrate the egg, most are degraded after a few hours. As the cells develop, more and more of the mtDNA from males is diluted out.  Hence less than one part in 104 or 0.01% of the mtDNA is paternal.

Mitochondria are largely maternally inherited in Mitochondria are largely maternally inherited in higher animals and plantshigher animals and plants

This means that mutations of mtDNA are passed from mother to child.  It also has implications for the cloning of mammals with the use of  somatic cells.  The nuclear DNA would be from the donor cell, but the mtDNA would be from the host cell.  This is how Dolly the sheep was cloned.  

In plants, the cytoplasm, including the mitochondria and the plastids, are contributed only by the female gamete and not by the pollen - again, mutations in organelle DNA are inherited maternally.

• Mitochondria divide by fission and are not made de novo

Human Evolution and mtDNAHuman Evolution and mtDNA

• they are inherited mainly from the mother: >99% of our mitochondria are derived from those (1000 or so) present in our mother’s ovum

Extrapolating this in evolutionary terms, this means that all mitochondria came from a “single” ancestral female

- the so-called “Mitochondrial Eve”.References:

Proceedings of National Academy Sci (USA) 91:8739 (1994)Science 279: 28 (1998)

However, this is based on the assumption that mitochondrial inheritance is strictly clonal. Recent evidence shows that mitos from sperm do enter the egg and last for several hours. If recombination occurs between mitos, then the Eve hypothesis may be incorrect - or at least the timing would be incorrect.

Proc. R. Soc. Lond. B (1999) 266, 477-483

D-loop: origin of mtDNA replication

Human evolution can be traced by analysis of the base sequence in a small part of the mitochondrial genome which does not encode a gene and which is quite variable.

- the so-called D-loop.

Human Evolution and mtDNAHuman Evolution and mtDNA

The D-Loop of the mtDNA is the start of replication/transcription site and contains 400-800 bp

Unlike the rest of mtDNA in humans, which is highly conserved, this region is very variable between people

It also has a very high frequency of change during evolution (about 2% per million years)

Human Evolution and mtDNAHuman Evolution and mtDNA

This makes the D-loop a very powerful tool for the study of evolutionary relationships between organisms and for DNA typing of individuals.

In addition, because of the large number of mitos in a cell, extracting mtDNA is easier from small amounts of tissue - and it can be readily separated form other DNA by centrifugation on CsCl gradients.

Human Evolution and mtDNAHuman Evolution and mtDNA

By comparing different groups, we can get a glimpse of human evolutionary lines.

Eg, African individuals have more variability between each other than do Asians, indicating that the former have had more time to accumulate changes - ie, the Africans are a more ancient group.

Human Evolution and mtDNAHuman Evolution and mtDNA

Assuming that the rate of change in the D-loop is constant and due only to mutation, the number of difference s between Africans can be use to calculate when their common ancestor lived. This works out to be about 200,000 years ago.

Human Evolution and mtDNAHuman Evolution and mtDNA

This suggests that modern Homo sapiens came out of Africa at about that time and migrated through Europe and Asia, replacing other early humans

But we have to be careful: the rate of change in mtDNA may not be constant and heteroplasmy (due to recombination of mtDNA) may cause complications. Also, mtDNA represents a single lineage and other genetic changes need to be traced also.

Human Evolution and mtDNAHuman Evolution and mtDNA

However, when this was done with polymorphisms in the Y chromosome, ‘Adam’ was also traced back to Africa, at about the same period.

What are Mitochondria - Evolution

Endosymbionts - Bacterium engulfed by precursor to Eukaryotic cells and formed a symbiotic relationship.

Gene Transfer - Accounts for the loss of mitochondrial genes to the nucleus.

Outstanding Questions:Are mitochondria simply ‘endosymbionts’ who have the majority of coding capacity in Host?Why aren’t all the genes transferred?

Rickettsia - 834 open reading frames (obligate intracellular parasite)

E. coli - 4, 288 ORFHuman mit genome - 13 ORFYeast mit genome - 7 ORFArabidopsis mit genome - 57 ORFReclinomonas americana - 67 ORF

These figures would suggest that mitochondria areEndosymbionts that have transferred most of their coding capacity to the host. However the process of gene transferwas (or is) not as straightforward as it may appear.

Gray et al. 2001

Yeast Mitochondrial Proteome

Classification Based on Phylogenetic Origin

Why aren’t all the genes transferred?

Rickettsia - 834 open reading framesE. coli - 4, 288 ORFHuman mit genome - 13 ORFYeast mit genome - 7 ORFArabidopsis mit genome - 57 ORFReclinomonas americana - 67 ORF

Hydrogenosomes are likely to be organelles that were mitochondria but have lost all DNA

Mitochondrial DNA of animals and fungi uses a different genetic code than the “universal” code

Mitochondrial gene

Nuclear gene

Integration and acquisition Of Nuclear Signals

Expression

Import and assembly

Dual Expression

DNA

RNA

DNA

RTGene Transfer

•Multi-step process

•Several potentialbarriers

Nucleus

Mitochondria

2. Gene aquires: (a) expression, and (b) mitochondrial targeting signals

1. Gene integrates into nuclear genome

Import of preprotein and

Intermembrane SpaceMatrix

MitochondrialTargeting

presequence cleavage

protein assembly

(a) (b)

3(a)

3(b)

45

Screening for Gene Transfer

Multiple transfersand activationmechanisms for aribosomal protein

cUQUQ1/2 O2H2OIIIIIVVF1FoNADHNAD+H+H+H+H+ADP + PiATPIISuccinateFumarateMatrixInner MembraneIntermembraneSpace

Apocytochrome bCOX 1

Genes Encoded in All Mitochondrial Genomes

COX Subunit Composition

Poyton and McEwen 1996

COX1preCOX2COX3

IMMOMMCOX4COX5ACOX5BCOX6COX7COX8COX9

Nucleus

Mitochondria

Gene Transfer of cox 2 in legumes

TM1TM2Matrix-loopN-loopC-loopA)16061124125136137161162181182202203228229376MTDTDMSSN-loopTM1Matrix-loopTM2C-loopPresequenceMature proteinB)Mitochondrial inner membraneIntermembrane spaceMatrix

Topology of Cox2 in Inner Mitochondrial Membrane

In vitro protein import into mitochondria

Nuclear-Mitochondrial Cox2 Chimerics

Organelle Encoded cox2 Cannot Be Imported

Transmembrane Region I of Mit Encoded cox2 Inhibits Import

<H>17 <H>19 Kyte and Doolittle aWW

Species Gene TM1 TM2 TM1 TM2

Soybean (Gm) mt 3.253 2.306 5.83 0.99n 2.559 2.288 3.82 1.78

Amphicarpea mt 3.524 2.306 6.52 0.98 bracteata (Ab) n 2.024 2.265 3.03 2.15

Dumasia mt 3.524 2.306 6.52 0.98 villosa n 2.729 2.306 2.96 1.77

Lespedeza mt 3.524 2.306 6.52 0.98 formosa n 1.888 2.265 1.87 0.90

Neonotonia mt 3.465 2.306 7.09 0.98 wightii n 2.712 2.306 2.92 1.49

Pseudeminia mt 3.524 2.306 6.52 0.98 comosa n 2.753 2.247 2.34 1.31

Hydrophobicity Changes in Transmembrane RegionsAssociated with cox2 Gene Transfer in legumes

A

1.5

2

2.5

3

3.5

4

0.75 1 1.25 1.5

mtGm

nGm

x

y

<H>60-80

nNw

nLf

nEpnAlnAb

nPc

nPenCc

nCu

nVu

nRs

nDvnOs

mtNw

mtAb, mtCc, mtCl, mtDv,mtLf, mtPa, mtPc, mtPe,mtTu

Hydrophobicity Verse Coding Locationof Cox2 from a Variety of Legume Species

B

1.5

2

2.53

3.5

4

0.75 1 1.25 1.5<H>

60-80

y

x

mtAt

mtHs

mtNc

nPs(a)

nCr(a)

mtSc

mtSkmtMm

mtBt

mtRn

Hydrophobicity Verse Coding Locationof Cox2 from a Variety of Species

Construct <H> 17 <H> 19 Import Kyte and Doolittle aWW

TM1 TM2 TM1 TM2

nGm 1-123/mGm 124-383 3.253 2.306 5.17 0.99 8

nGm1-123/mGm124-383 L169Q 2.824 2.306 4.03 0.99 8

nGm1-123/mGm124-383 L169Q/L171G 2.576 2.306 3.46 0.99 4

nGmCox2 2.559 2.288 3.16 1.78 4

nGmCox2 2.559 2.288 3.16 1.78 4

nGmCox2 Q169L 2.988 2.288 4.30 1.78 4

nGmCox2 Q169L/G171L 3.235 2.288 4.87 1.78 8

nGm1-123/mGm124-383 3.253 2.306 5.17 0.99 8

Amino Acid Changes that Reduce Hydrophobicity in Mature Protein Required for Import

+

_

+

_

+

+_

_

Hydrophobicity is a barrier to import and thus Gene Transfer - but only for some genes.

In non-plant systems Hydrophobicity should not be a problem - non-universal genetic code is the barrier.

This implies in plants some other mechanism(s) operating to maintain mitochondrial genome.

Why aren’t all the genes transferred?

Chloroplast

Mitochondria

Photosystem II Cytochrome b /f6 Photosystem I ATP synthase

RuBisCORibosome

Stroma

Lumen

Complex I Complex II Complex III Complex V

Ribosome

Intermembrane Space

Matrix

Complex IV

Missing Ribosomal ProteinsGenes for rps 8 and 13 of the mitochondrial ribosome missing fromArabidopsis nuclear and mitochondrial genomes.

These genes are considered essential No ESTHard to envisage how ribosome can function without these proteins - experimental evidence indicatesimpossible

Phylogenetic Analysis ofMitochondrial and Chloroplastrps13 Genes

In vitro Mitochondrial and Chloroplast Import Assays

plant cDNA transcription & translation cell-free lysate mitochondria room temp 20 min 25˚C 25 min Divide into 2 equal aliquots precursor alone precursor + mito precursor + mito + protease protease Phosphor imaging of polyacrylamide gel Precursor Mature form Precursor Mature chloroplasts + proteinase K + thermolysin + chloro + chloro incubation [ S] labelled precursor protein

35

Duplicated rps13 is Targeted to Mitochondria

But not Chloroplasts

Phylogenetic Analysis ofrps15a Genes

Missing Ribosomal Proteins

Current mitochondrial proteome has a complexgenetic history.

In this case the Arabidopsis mitochondrial ribosomeis derived from at least three different ancestors.

Replaced

Why aren’t all the genes transferred?

Hydrogenosomes are likely to be organelles that were mitochondria but have lost all DNA

All Plant mitpchondrial Genomes encode some ribosomal Proteins - not hydrophobic

Assembly - All organelle encoded proteins function inMultisubunit complexes. Defined and sequential assemblyPathways may dictate some proteins encoded.

Ribosomes are very complex and have very specific Assembly pathways