Plant Energy Biology

2. Plant Energy Biology New knowledge research…

3. 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

4. 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

5. 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

6. 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

7. 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

8. Mitochondrial molecular biology 2 • evolution of mitochondria • maternal inheritance of mtDNA • mtDNA and human evolution

9. 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)

10. Organisation of the mitochondrial chromosome 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.

11. Yeast mitochondrial chromosome yeast mtDNA • 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 human mtDNA

12. cell division: random distribution of mitos between daughter cells mitochondrial replication Mitochondrial replication 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.

13. 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.

14. Endosymbiotic hypothesis of mitochondrial evolution

15. Evolution of mitochondria 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.

16. endocytosis host membrane 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.

17. Chloroplasts of plants and algae are thought to have arisen from endosymbiosis of a cyanobacterium (blue-green alga)

18. 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.

19. 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.

20. 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.

21. 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.

22. 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.

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

24. Synthesis of mitochondrial proteins 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.

25. Mitochondria are largely maternally inherited in higher animals 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.

26. Mitochondria are largely maternally inherited in higher 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.

27. Human Evolution and mtDNA • Mitochondria divide by fission and are not made de novo • they are inherited mainly from the mother: >99% of our mitochondria are derived from those (1000 or so) present in our mother’s ovum

28. 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

29. D-loop: origin of mtDNA replication Human Evolution and mtDNA 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.

30. Human 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)

31. Human 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.

32. Human 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.

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

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

35. 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?

36. Rickettsia - 834 open reading frames (obligate intracellular parasite) E. coli - 4, 288 ORF Human mit genome - 13 ORF Yeast mit genome - 7 ORF Arabidopsis mit genome - 57 ORF Reclinomonas americana - 67 ORF These figures would suggest that mitochondria are Endosymbionts that have transferred most of their coding capacity to the host. However the process of gene transfer was (or is) not as straightforward as it may appear.

37. Yeast Mitochondrial Proteome Classification Based on Phylogenetic Origin Gray et al. 2001

38. Why aren’t all the genes transferred? Rickettsia - 834 open reading frames E. coli - 4, 288 ORF Human mit genome - 13 ORF Yeast mit genome - 7 ORF Arabidopsis mit genome - 57 ORF Reclinomonas americana - 67 ORF Hydrogenosomes are likely to be organelles that were mitochondria but have lost all DNA

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

40. Mitochondrial gene RNA RT DNA DNA Integration and acquisition Of Nuclear Signals Expression Import and assembly Dual Expression Nuclear gene • Gene Transfer • Multi-step process • Several potential • barriers

42. Screening for Gene Transfer

43. Multiple transfers and activation mechanisms for a ribosomal protein

44. Genes Encoded in All Mitochondrial Genomes COX 1 Apocytochrome b

45. COX Subunit Composition Mitochondria Poyton and McEwen 1996

46. Gene Transfer of cox 2 in legumes

47. Topology of Cox2 in Inner Mitochondrial Membrane

49. In vitro protein import into mitochondria