Slide 1Chloroplast BiologySlide 3Slide 4Slide 5Slide 6Slide 7Slide 8III. ReproductionIV. DevelopmentSlide 11Slide 12Slide 13Slide 14Slide 15V. Chloroplast GeneticsSlide 17Slide 18Slide 19Chloroplast DNA (cpDNA)Slide 21Pioneers in chloroplast & plant molecular biologyDiscovered chloroplast genetics (Chlamydomonas) and DNA Chloroplast genes in corn; demonstrated light regulationChloroplast gene function; nuclear control Ruth Sager Lawrence Bogorad Jean-David Rochaix (Harvard) (Harvard) (Geneva)Chloroplast BiologyI. Structure1. double-membrane envelope2. stroma: large soluble interior3. thylakoid membrane system4. intrathylakoid space or lumenFrom Gunning & SteerIsolated Spinach chloroplastenvelopestromathylakoid membraneFrom HooberHigh magnification of thylakoid membranes From HooberStroma of Avena (rye) plastid at high magnification to show ribosomesS – aggregates of a glucosidase used in defense against fungi, substrate is in vacuole (avenacoside), product is a toxic saponinFrom Gunning & SteerII. Functions1. many important biochemical (anabolic) pathways, e.g., photosynthesis*starch synthesis fatty acid synthesis*amino acids synthesispigment synthesis*nucleotide synthesisnucleic acids* and protein synthesissulfur and nitrogen assimilation2. own genetic system* * Indicates that pathway involves a chloroplast encoded gene in at least some organismsMany 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)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 dividingFrom Miyagishima et al.IV. DevelopmentThere are several forms of plastids:1. Proplastids - precursor of all plastids, found in meristems2. Etioplasts - form in shoots of dark-grown plants, distinctive internal structure3. Chloroplasts - in all green tissues4. Amyloplasts - prominent in roots, store starch, colorless5. Chromoplasts - in mature fruit, lots of carotenoids, little chlorophyllMature EtioplastProplastidPlastid development in dark-grown barleyYoungestOldestAmyloplasts from Glycine (soybean) root-cap (peripheral cell)S - starch grainsAlso have some thylakoid membranes (star) close-by.From Gunning & SteerFrom U. Wisconsin Botany Dept.Young chromoplast from developing tomato fruitFrom Gunning and Steer Stars mark lycopene crystals; many plastoglobuliPlastid development is plastic & mostly under nuclear control.Shoots: lightproplastids etioplasts chloroplasts chromoplasts Roots:proplastids amyloplastsV. Chloroplast Genetics1. 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 cell2. 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.Chlamydomonas life cycle has sexual and asexual reproduction.For sex, there are 2 mating types, mt+ and mt-, mt- cpDNA destroyedNishimura, Yoshiki et al. (1999) Proc. Natl. Acad. Sci. USA 96, 12577-12582Digestion of cpDNA of the mt- parent in a young zygote of Chlamydomonas revealed by fluorescence staining of DNA.0 Minutes10 minutesFrom Mauseth, 1998Acetabularia – green alga, fossils known, many species likely extinctA single giant cell (5 cm), 1 nucleus, ~1 x 106 chloroplasts~30 % of chloroplasts don’t have DNA!nucleusChloroplast DNA (cpDNA)General features:1. double-stranded, circular molecule2. no histones, but have bound proteins (e.g., Hu), organized into nucleoids3. G-C content typically less than nuclear DNA 4. multiple copies (~30-100) per plastid (i.e., all cp genes are multi-copy)5. can be 10-20% of the total DNA in leaves“relaxed” cpDNA molecule from lettuceFrom Kolodner &