Pioneers in chloroplast & plant molecular biology

1. Pioneers in chloroplast & plant molecular biology Ruth Sager Lawrence Bogorad Jean-David Rochaix (Harvard) (Harvard) (Geneva) Discovered chloroplast genetics (Chlamydomonas) and DNA Chloroplast genes in corn; demonstrated light regulation Chloroplast gene function; nuclear control

2. Chloroplast Biology I. Structure • double-membrane envelope • stroma: large soluble interior • thylakoid membrane system • intrathylakoid space or lumen

3. From Gunning & Steer

4. Isolated Spinach chloroplast envelope thylakoid membrane stroma From Hoober

5. High magnification of thylakoid membranes From Hoober

6. Stroma of Avena (rye) plastid at high magnification to show ribosomes S – aggregates of a glucosidase used in defense against fungi, substrate is in vacuole (avenacoside), product is a toxic saponin From Gunning & Steer

7. II. Functions many important biochemical (anabolic) pathways, e.g., photosynthesis* starch synthesis fatty acid synthesis* amino acids synthesis pigment synthesis* nucleotide synthesis nucleic acids* and protein synthesis sulfur and nitrogen assimilation 2. own genetic system* * Indicates that pathway involves a chloroplast encoded gene in at least some organisms

8. Many of the biosynthetic pathways are regulated, to peak during the light period of L-D cycle. Chlorophyll a and b synthesis during a 24 hour light-dark cycle (Chlamydomonas). A similar result was obtained for carotenoids and other chloroplast lipids. (Janero and Barnett, 1982)

9. III. Reproduction • all plant and eukaryotic algal cells have plastids • chloroplasts form by division; semi-autonomous • Involves proteins (Fts) similar to those that mediate cell division in bacteria Cyanidioschyzon chloroplast dividing From Miyagishima et al.

10. IV. Development There are several forms of plastids: 1. Proplastids - precursor of all plastids, found in meristems 2. Etioplasts - form in shoots of dark-grown plants, distinctive internal structure 3. Chloroplasts - in all green tissues 4. Amyloplasts - prominent in roots, store starch, colorless 5. Chromoplasts - in mature fruit, lots of carotenoids, little chlorophyll

11. Plastid development in dark-grown barley Mature Etioplast Oldest Youngest Proplastid

12. Amyloplasts from Glycine (soybean) root-cap (peripheral cell) S - starch grains Also have some thylakoid membranes (star) close-by. From Gunning & Steer

13. From U. Wisconsin Botany Dept.

14. Young chromoplast from developing tomato fruit Stars mark lycopene crystals; many plastoglobuli From Gunning and Steer

15. Plastid development is plastic & mostly under nuclear control. Shoots: light proplastids etioplasts chloroplasts chromoplasts Roots: proplastids amyloplasts

16. V. Chloroplast Genetics 1. Inheritance is typically uniparental, usually maternal. Multiple mechanisms involved, not well understood: - in Chlamydomonas (next slide), the paternal (-) cpDNA is destroyed, and the maternal (+) cpDNA is preferentially replicated - in some land plants, the paternal plastids are excluded during fertilization or absent from the sperm cell 2. Essentially all plastids have DNA, usually the same DNA throughout the organism (homoplasmy). 3. The DNA sequence does not change during differentiation. There are exceptions to the last 2 statements.

17. Chlamydomonas life cycle has sexual and asexual reproduction. For sex, there are 2 mating types, mt+ and mt-, mt- cpDNA destroyed

18. Digestion of cpDNA of the mt- parent in a young zygote of Chlamydomonas revealed by fluorescence staining of DNA. 0 Minutes 10 minutes Nishimura, Yoshiki et al. (1999) Proc. Natl. Acad. Sci. USA 96, 12577-12582

19. From Mauseth, 1998 Acetabularia – green alga, fossils known, many species likely extinct A single giant cell (5 cm), 1 nucleus, ~1 x 106 chloroplasts ~30 % of chloroplasts don’t have DNA! nucleus

20. Chloroplast DNA (cpDNA) General features: • double-stranded, circular molecule • no histones, but have bound proteins (e.g., Hu), organized into nucleoids • G-C content typically less than nuclear DNA • multiple copies (~30-100) per plastid (i.e., all cp genes are multi-copy) • can be 10-20% of the total DNA in leaves

21. “relaxed” cpDNA molecule from lettuce From Kolodner & Tewari